Matrix Metalloproteinase Substrates And Other Cleavable Moieties And Methods Of Use Thereof

ABSTRACT

The invention relates generally to polypeptides that include a cleavable moiety that is a substrate for at least one matrix metalloprotease (MMP), to activatable antibodies and other larger molecules that include the cleavable moiety that is a substrate for at least one MMP protease, and to methods of making and using these polypeptides that include a cleavable moiety that is a substrate for at least one MMP protease in a variety of therapeutic, diagnostic and prophylactic indications.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/882,377, filed Sep. 25, 2013 and U.S. Provisional Application No.61/971,332, filed Mar. 27, 2014, the contents of each of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates generally to polypeptides that include a cleavablemoiety that is a substrate for at least one matrix metalloprotease(MMP), to activatable antibodies and other larger molecules that includethe cleavable moiety that is a substrate for at least one MMP protease,and to methods of making and using these polypeptides that include acleavable moiety that is a substrate for at least one MMP protease in avariety of therapeutic, diagnostic and prophylactic indications.

BACKGROUND OF THE INVENTION

Proteases are enzymes that degrade proteins by cleaving the peptidebonds between amino acid residues. Proteases occur naturally in allorganisms and are involved in a variety of physiological reactions fromsimple degradation to highly regulated pathways. Some proteases areknown to break specific peptide bonds based on the presence of aparticular amino acid sequence within a protein.

Accordingly, there exists a need to identify new substrates forproteases and to use these substrates in a variety of therapeutic,diagnostic and prophylactic indications.

SUMMARY OF THE INVENTION

The disclosure provides amino acid sequences that include a cleavablemoiety (CM) that is a substrate for at least one matrix metalloprotease(MMP). These CMs are useful in a variety of therapeutic, diagnostic andprophylactic indications.

In some embodiments, the CM is a substrate for at least one matrixmetalloprotease (MMP). Examples of MMPs include MMP1; MMP2; MMP3; MMP7;MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17;MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. In some embodiments, theCM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, andMMP19. In some embodiments, the CM is a substrate for MMP9. In someembodiments, the CM is a substrate for MMP14. In some embodiments, theCM is a substrate for two or more MMPs. In some embodiments, the CM is asubstrate for at least MMP9 and MMP14. In some embodiments, the CMcomprises two or more substrates for the same MMP. In some embodiments,the CM comprises at least two or more MMP9 substrates. In someembodiments, the CM comprises at least two or more MMP14 substrates.

In some embodiments, the CM is a substrate for an MMP and includes thesequence ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID NO: 15); AQNLLGMV(SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17); PVGYTSSL (SEQ ID NO: 18);DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ ID NO: 20); RPSPMWAY (SEQ ID NO:21); WATPRPMR (SEQ ID NO: 22); FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQID NO: 24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG(SEQ ID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29);LAAPLGLL (SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO:32); PAGLWLDP (SEQ ID NO: 33); and/or ISSGLSS (SEQ ID NO: 159).

In some embodiments, the CM comprises the amino acid sequence ISSGLLSS(SEQ ID NO: 14). In some embodiments, the CM comprises the amino acidsequence QNQALRMA (SEQ ID NO: 15). In some embodiments, the CM comprisesthe amino acid sequence AQNLLGMV (SEQ ID NO: 16). In some embodiments,the CM comprises the amino acid sequence STFPFGMF (SEQ ID NO: 17). Insome embodiments, the CM comprises the amino acid sequence PVGYTSSL (SEQID NO: 18). In some embodiments, the CM comprises the amino acidsequence DWLYWPGI (SEQ ID NO: 19). In some embodiments, the CM comprisesthe amino acid sequence MIAPVAYR (SEQ ID NO: 20). In some embodiments,the CM comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 21). Insome embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQID NO: 22). In some embodiments, the CM comprises the amino acidsequence FRLLDWQW (SEQ ID NO: 23). In some embodiments, the CM comprisesthe amino acid sequence LKAAPRWA (SEQ ID NO: 24). In some embodiments,the CM comprises the amino acid sequence GPSHLVLT (SEQ ID NO: 25). Insome embodiments, the CM comprises the amino acid sequence LPGGLSPW (SEQID NO: 26). In some embodiments, the CM comprises the amino acidsequence MGLFSEAG (SEQ ID NO: 27). In some embodiments, the CM comprisesthe amino acid sequence SPLPLRVP (SEQ ID NO: 28). In some embodiments,the CM comprises the amino acid sequence RMHLRSLG (SEQ ID NO: 29). Insome embodiments, the CM comprises the amino acid sequence LAAPLGLL (SEQID NO: 30). In some embodiments, the CM comprises the amino acidsequence AVGLLAPP (SEQ ID NO: 31). In some embodiments, the CM comprisesthe amino acid sequence LLAPSHRA (SEQ ID NO: 32). In some embodiments,the CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 33). Insome embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQID NO: 159).

In some embodiments, the CM is linked or otherwise attached to anantibody. For example, the CM is used to link one or more agents to theantibody or antigen binding fragment thereof (AB) that binds a giventarget, such that the CM is cleaved when exposed to the MMP and theagent is released from the AB. Exemplary targets include, but are notlimited to the targets shown in Table 1. Exemplary ABs include, but arenot limited to, the targets shown in Table 2. In some embodiments, theantibody in the uncleaved state has the structural arrangement fromN-terminus to C-terminus as follows: Agent-CM-AB or AB-CM-Agent. In someembodiments, the antibody comprises a linking peptide between the AB andthe CM. In some embodiments, the antibody comprises a linking peptidebetween the CM and the conjugated agent.

In some embodiments, the antibody comprises a first linking peptide(LP1) and a second linking peptide (LP2), and the antibody in theuncleaved state has the structural arrangement from N-terminus toC-terminus as follows: Agent-LP1-CM-LP2-AB or AB-LP2-CM-LP1-Agent. Insome embodiments, each of LP1 and LP2 is a peptide of about 1 to 20amino acids in length. In some embodiments, the two linking peptidesneed not be identical to each other.

In some embodiments, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 1) and (GGGS)_(n) (SEQ ID NO: 2), where n is aninteger of at least one.

In some embodiments, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of GGSG (SEQ ID NO: 3),GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG(SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8).

In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG(SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID NO:11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO: 156), orGSSGGSGGSGS (SEQ ID NO: 157).

In some embodiments, LP2 comprises the amino acid sequence GSS, GGS,GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).

In some embodiments, the AB has an equilibrium dissociation constant ofabout 100 nM or less for binding to the target.

In some embodiments, the antibody includes an antibody orantigen-binding fragment thereof that specifically binds a target. Insome embodiments, the antibody or immunologically active fragmentthereof that binds the target is a monoclonal antibody, domain antibody,single chain, Fab fragment, a F(ab′)₂ fragment, a scFv, a scab, a dAb, asingle domain heavy chain antibody, or a single domain light chainantibody. In some embodiments, such an antibody or immunologicallyactive fragment thereof that binds the target is a mouse, other rodent,chimeric, humanized or fully human monoclonal antibody.

In some embodiments, the MMP protease is co-localized with the target ina tissue, and the MMP protease cleaves the CM in the antibody when theantibody is exposed to the protease.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength.

In some embodiments, the CM is a substrate for at least one matrixmetalloprotease (MMP). Examples of MMPs include MMP1; MMP2; MMP3; MMP7;MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17;MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. In some embodiments, theCM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, andMMP19. In some embodiments, the CM is a substrate for MMP9. In someembodiments, the CM is a substrate for MMP14. In some embodiments, theCM is a substrate for two or more MMPs. In some embodiments, the CM is asubstrate for at least MMP9 and MMP14. In some embodiments, the CMcomprises two or more substrates for the same MMP. In some embodiments,the CM comprises at least two or more MMP9 substrates. In someembodiments, the CM comprises at least two or more MMP14 substrates.

In some embodiments, the CM is a substrate for an MMP and includes thesequence ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID NO: 15); AQNLLGMV(SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17); PVGYTSSL (SEQ ID NO: 18);DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ ID NO: 20); RPSPMWAY (SEQ ID NO:21); WATPRPMR (SEQ ID NO: 22); FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQID NO: 24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG(SEQ ID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29);LAAPLGLL (SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO:32); PAGLWLDP (SEQ ID NO: 33); and/or ISSGLSS (SEQ ID NO: 159).

In some embodiments, the CM comprises the amino acid sequence ISSGLLSS(SEQ ID NO: 14). In some embodiments, the CM comprises the amino acidsequence QNQALRMA (SEQ ID NO: 15). In some embodiments, the CM comprisesthe amino acid sequence AQNLLGMV (SEQ ID NO: 16). In some embodiments,the CM comprises the amino acid sequence STFPFGMF (SEQ ID NO: 17). Insome embodiments, the CM comprises the amino acid sequence PVGYTSSL (SEQID NO: 18). In some embodiments, the CM comprises the amino acidsequence DWLYWPGI (SEQ ID NO: 19). In some embodiments, the CM comprisesthe amino acid sequence MIAPVAYR (SEQ ID NO: 20). In some embodiments,the CM comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 21). Insome embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQID NO: 22). In some embodiments, the CM comprises the amino acidsequence FRLLDWQW (SEQ ID NO: 23). In some embodiments, the CM comprisesthe amino acid sequence LKAAPRWA (SEQ ID NO: 24). In some embodiments,the CM comprises the amino acid sequence GPSHLVLT (SEQ ID NO: 25). Insome embodiments, the CM comprises the amino acid sequence LPGGLSPW (SEQID NO: 26). In some embodiments, the CM comprises the amino acidsequence MGLFSEAG (SEQ ID NO: 27). In some embodiments, the CM comprisesthe amino acid sequence SPLPLRVP (SEQ ID NO: 28). In some embodiments,the CM comprises the amino acid sequence RMHLRSLG (SEQ ID NO: 29). Insome embodiments, the CM comprises the amino acid sequence LAAPLGLL (SEQID NO: 30). In some embodiments, the CM comprises the amino acidsequence AVGLLAPP (SEQ ID NO: 31). In some embodiments, the CM comprisesthe amino acid sequence LLAPSHRA (SEQ ID NO: 32). In some embodiments,the CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 33). Insome embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQID NO: 159).

In some embodiments, the CM is a substrate for at least one matrixmetalloprotease (MMP) and includes a motif sequence that is recognizedby MMP9. In some embodiments, the CM is a substrate for at least one MMPand includes a motif sequence that is recognized by MMP14.

In some embodiments, the CM is a substrate for at least one MMP, and theCM polypeptide and/or the CM portion of any polypeptide that comprisesthe CM comprises a polypeptide having a length less than 50 amino acids,less than 40 amino acids, less than 30 amino acids, less than 25 aminoacids, less than 20 amino acids, less than 19 amino acids, less than 18amino acids, less than 17 amino acids, less than 16 amino acids, lessthan 15 amino acids long, less than 14 amino acids, less than 13 aminoacids, less than 12 amino acids, less than 11 amino acids, or less than10 amino acids long.

In some embodiments, the CM is a substrate for at least one MMP andcomprises a polypeptide sequence that is not substantially identical toany human polypeptide sequence that is naturally cleaved by the same MMPprotease. In some embodiments, the CM is a substrate for at least oneMMP and comprises a polypeptide sequence that is no more than 90% ormore identical to any human polypeptide sequence that is naturallycleaved by the same MMP protease.

In some embodiments, the motif sequence is a substrate for at least MMPand includes a core CM consensus sequence shown in Tables 8A-8M below.In some embodiments, the motif sequence includes a subgenus, i.e., asubset, of the core CM consensus sequence shown in Tables 8A-8M below.

In some embodiments, the motif sequence is a substrate for at least MMP9and includes a core CM consensus sequence shown in Tables 8A-8D. In someembodiments, the motif sequence is a substrate for at least MMP9 andincludes a subgenus, i.e., a subset, of the core CM consensus sequenceshown in Tables 8A-8D below.

In some embodiments, the motif sequence is a substrate for at leastMMP14 and includes a core CM consensus sequence shown in Tables 8E-8M.In some embodiments, the motif sequence is a substrate for at leastMMP14 and includes a subgenus, i.e., a subset, of the core CM consensussequence shown in Tables 8E-8M below.

TABLE 8A MMP9 Cleavable Core CM Consensus Sequence 1 Core CM Consensus 1Subgenus of Core CM Consensus 1 X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉ Subgenus 1.1:X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉ (SEQ ID NO: 318), (SEQ ID NO: 317), whereinwherein X₂₂ is G, P, R, or S; X₂₃ is P or S; X₂₄ is L, M, P, or S; X₂₂is A, C, D, G, H, L, P, X₂₅ is A, G, P, or S; X₂₆ is L, M, or R; X₂₇ isG or W; X₂₈ is A, R, or S; G, S, or Y; and X₂₉ is L, R, V, or Y. X₂₃ isL, M, P, S, or T; Subgenus 1.2: X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉ (SEQ ID NO:319), X₂₄ is A, D, F, G, L, M, N, wherein X₂₂ is G, P or R; X₂₃ is P;X₂₄ is L, M, or S; X₂₅ is G, P, R, S, T, or V; P, or S; X₂₆ is L, M, orR; X₂₇ is W; X₂₈ is A, G, or S; and X₂₉ X₂₅ is A, D, E, G, H, I, M, isR, V, or Y. P, S, or V; Subgenus 1.3: X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉ (SEQ IDNO: 320), X₂₆ is A, C, D, G, L, M, N, wherein X₂₂ is P or R; X₂₃ is P;X₂₄ is M or S; X₂₅ is G or P; X₂₆ R, V, W, or Y; is L, M, or R; X₂₇ isW; X₂₈ is A, G, or S; and X₂₉ is R, V, or Y. X₂₇ is C, F, G, H, P, Q,Subgenus 1.4: X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉ (SEQ ID NO: 321), R, T, V, or W;wherein X₂₂ is P or R; X₂₃ is P; X₂₄ is S; X₂₅ is G or P; X₂₆ is X₂₈ isA, D, G, L, M, S, T, M, or R; X₂₇ is W; X₂₈ is A or S; and X₂₉ is Y. V,or Y; and Subgenus 1.5: X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉ (SEQ ID NO: 322), X₂₉is C, H, L, R, S, V, W, wherein X₂₂ is P or R; X₂₃ is P; X₂₄ is S; X₂₅is G or P; X₂₆ is or Y. M, or R; X₂₇ is W; X₂₈ is A or S; and X₂₉ is Y.Subgenus 1.6: X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉ (SEQ ID NO: 323), wherein X₂₂ isC, G, H, L, or R; X₂₃ is P, S or T; X₂₄ is N, R, S or T; X₂₅ is P or S;X₂₆ is C, M, R, V, or W; X₂₇ is C, P, R, or W; X₂₈ is A, D, or G; andX₂₉ is C or Y.

TABLE 8B MMP9 Cleavable Core CM Consensus Sequence 2 Core CM Consensus 2Subgenus of Core CM Consensus 2 X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉ Subgenus 2.1:X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉ (SEQ ID (SEQ ID NO: 324), wherein NO: 325),wherein X₃₂ is W; X₃₃ is D, P, or T; X₃₄ is H, Q, X₃₂ is F, G, V, or W;or W; X₃₅ is D or P; X₃₆ is I or R; X₃₇ is S; X₃₈ is L, X₃₃ is A, D, L,M, P, R, T, or M, or V; and X₃₉ is G, L, or S. V; Subgenus 2.2:X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉ (SEQ ID X₃₄ is C, G, H, L, Q, S, T, W, NO:326), wherein X₃₂ is W; X₃₃ is D; X₃₄ is H, Q, or W; X₃₅ X₃₅ is D, G, L,P; is D or P; X₃₆ is I or R; X₃₇ is G, S, or V; X₃₈ is L, M, X₃₆ is E,G, I, L, N, P, R, or V; or V; and X₃₉ is G, L, or S. X₃₇ is G, L, P, R,S, or V; Subgenus 2.3: X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉ (SEQ ID X₃₈ is A, I, L,M, T, or V; and NO: 327), wherein X₃₂ is W; X₃₃ is D; X₃₄ is H, Q, or W;X₃₅ X₃₉ is A, G, L, P, Q, R, S, or V. is P; X₃₆ is I or R; X₃₇ is S; X₃₈is L, M, or V; and X₃₉ is L.

TABLE 8C MMP9 Cleavable Core CM Consensus Sequence 3 Core CM Consensus 3Subgenus of Core CM Consensus 3 X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉ Subgenus 3.1:X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉ (SEQ ID NO: 330), (SEQ ID NO: 329), wherein X₄₂is I, L, M, or S; X₄₃ is D, P, S, or T; X₄₄ is F, L, S, or wherein V;X₄₅ is L. P, or S; X₄₆ is A, F, R, S, or T; X₄₇ is G, H, T or Y; X₄₂ isG, I, L, M, P, R, S, X₄₈ is G, I, M, V, or W; and X₄₉ is F, L, or S. T,or V; Subgenus 3.2: X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉ (SEQ ID NO: 331), X₄₃ is A,D, H, I, L, P, S, wherein X₄₂ is L, M, or S; X₄₃ is S or T; X₄₄ is F orL; X₄₅ is P; X₄₆ or T; is A, F, or T; X₄₇ is G, H, T or Y; X₄₈ is I, M,or W; and X₄₉ is F. X₄₄ is F, L, S, or V; Subgenus 3.3:X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉ (SEQ ID NO: 332), X₄₅ is H, L, M, P, Q, R,wherein X₄₂ is L, M, or S; X₄₃ is S or T; X₄₄ is F; X₄₅ is P; X₄₆ is S,or T; A, F, or T; X₄₇ is G, H, or Y; X₄₈ is I, M, or W; and X₄₉ is F.X₄₆ is A, D, F, G, L, M, Subgenus 3.4: X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉ (SEQ IDNO: 333), R, S, T, or V; wherein X₄₂ is L or M; X₄₃ is S or T; X₄₄ is F;X₄₅ is P; X₄₆ is A or T; X₄₇ is A, C, G, H, Q, T or X₄₇ is H or Y; X₄₈is I or W; and X₄₉ is F. Y; Subgenus 3.5: X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉ (SEQID NO: 334), X₄₈ is C, G, I, M, R, S, T, wherein X₄₂ is G, I, R, Or S;X₄₃ is H Or T; X₄₄ is F, L, S, or V; X₄₅ V, or W; and is L, P, or R; X₄₆is F, L, or S; X₄₇ is A, C, or G; X₄₈ is I, M, or V; and X₄₉ is F, L, S,or Y. X₄₉ is F or L. Subgenus 3.6: X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉ (SEQ ID NO:335), wherein X₄₂ is S; X₄₃ is T; X₄₄ is F or V; X₄₅ is L or P; X₄₆ is For L; X₄₇ is G; X₄₈ is I or M; and X₄₉ is F.

TABLE 8D MMP9 Cleavable Core CM Consensus Sequence 4 Core CM Consensus 4Subgenus of Core CM Consensus 4 X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈X₅₉ Subgenus 4.1:X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈X₅₉ (SEQ ID NO: 341), (SEQ ID NO: 340), wherein X₅₂is D, G, H, L, P, Q, S or Y X₅₃ is D, W, or Y; X₅₄ is wherein H, L, orR; X₅₅ is H, L, M, P, or Y; X₅₆ Is E, F, G, M, R, or W; X₅₇ X₅₂ is D, G,H, L, N, P, is A, L, M, N, P, or R; X₅₈ is G, L, P, R, or S; and X₅₉ isG, I, P, S, T, Q, R, S, W, or Y or Y. X₅₃ is A, C, D, G, L, R, Subgenus4.2: X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈X₅₉ (SEQ ID NO: 342), V, W, or Y; wherein X₅₂is D or H; X₅₃ is W or Y; X₅₄ is H or L; X₅₅ is H, L, X₅₄ is D, H, L, P,Q, R, S, or Y; X₅₆ IS G or W; X₅₇ is P or R; X₅₈ is G, L, or P; and X₅₉is G, or Y; I, S, or T. X₅₅ is D, F, H, I, L, M, P, Subgenus 4.3:X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈X₅₉ (SEQ ID NO: 343), S, or Y; wherein X₅₂ is H;X₅₃ is W; X₅₄ is H or L; X₅₅ is H, L, or Y; X₅₆ Is X₅₆ is A, C, E, F, G,K, G or W; X₅₇ is P; X₅₈ is L or P; and X₅₉ is G, I, S or T. M, R, S, V,or W; Subgenus 4.4: X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈X₅₉ (SEQ ID NO: 344), X₅₇ is A,G, K L, M, N, wherein X₅₂ is H; X₅₃ is W; X₅₄ is H or L; X₅₅ is L or Y;X₅₆ Is G; P, R, S, or T; X₅₇ is P; X₅₈ is L or P; and X₅₉ is G, I, S, orT. X₅₈ is A, F, G, H, L, P, R, Subgenus 4.5: X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈X₅₉(SEQ ID NO: 345), S, or T; and wherein X₅₂ is H; X₅₃ is W; X₅₄ is H or LX₅₅ is L or Y X₅₆ Is G; X₅₉ is A, G, H, I, N, P, S, X₅₇ is P; X₅₈ is P;and X₅₉ is T. T, or Y. Subgenus 4.6: X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈X₅₉ (SEQ IDNO: 346), wherein X₅₂ is D, G, S, or Y; X₅₃ is W; X₅₄ is L or P; X₅₅ isD or Y; X₅₆ is C, E, G, or W; X₅₇ is M or P; X₅₈ is G, R, or S; and X₅₉is H, I, or Y. Subgenus 4.7: X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈X₅₉ (SEQ ID NO: 347),wherein X₅₂ is D, G, or S; X₅₃ is W; X₅₄ is L; X₅₅ is Y; X₅₆ is E or W;X₅₇ is M or P; X₅₈ is G or S; and X₅₉ is I or Y.

TABLE 8E MMP14 Cleavable Core CM Consensus Sequence 5 Core CM Consensus5 Subgenus of Core CM Consensus 5 X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ Subgenus 5.1:X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ (SEQ ID NO: 353), (SEQ ID NO: 352), whereinwherein X₆₂ is A, G, I, P, Q, S, T, or V; X₆₃ is A, L, Q, S, or V; X₆₂is A, I, G, L, M, P, Q, X₆₄ is A, E, L, R, or S; X₆₅ is D or G; X₆₆ is Ior L; X₆₇ is E, I, L, S, T, or V; M, Q, R, or Y; X₆₈ is F, H, L, M, R,or S; and X₆₉ is A, G, H, L, X₆₃ is A, D, L, P, Q, S, T, N, P, Q, or S.V, or Y; Subgenus 5.2: X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ (SEQ ID NO: 354), X₆₄ isA, C, E, F, G, H, K, wherein X₆₂ is A, I, S or T; X₆₃ is L, Q, S, or V;X₆₄ is A, L, R, L, P, Q, R, S, or V; or S; X₆₅ is G; X₆₆ is I or L; X₆₇is E, L, R, or Y; X₆₈ is F, H, L, R, or X₆₅ is D, E, G, S, or V; S; andX₆₉ is H, L, P, or S. X₆₆ is A, I, L, M, or V; Subgenus 5.3:X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ (SEQ ID NO: 355), X₆₇ is C, E, G, I, K, L, M,wherein X₆₂ is A, I, S or T; X₆₃ is L, S, or V; X₆₄ is A, R or S; N, Q,R, or Y; X₆₅ is G; X₆₆ is L; X₆₇ is E, L or R; X₆₈ is F, H, or S; andX₆₉ is X₆₈ is A, F, H, I, L, M, N, L, P, or S. P, R, S, or T; andSubgenus 5.4: X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ (SEQ ID NO: 356), X₆₉ is A, C, G,H, I, L, N, wherein X₆₂ is A, I, S or T; X₆₃ is L, S, or V; X₆₄ is R orS; X₆₅ is P, Q, R, S, T, V, or W. G; X₆₆ is L; X₆₇ is L or R; X₆₈ is F,H, or S; and X₆₉ is P or S. Subgenus 5.5: X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ (SEQID NO: 357), wherein X₆₂ is A, I, S or T; X₆₃ is L, S, or V;X_(64 is R or S; X) ₆₅ is G; X₆₆ is L; X₆₇ is L or R; X₆₈ is S; and X₆₉is P or S. Subgenus 5.6: X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ (SEQ ID NO: 358),wherein X₆₂ is T; X₆₃ is L, S, or V; X₆₄ is S; X₆₅ is G; X₆₆ is L; X₆₇is R; X₆₈ is S; and X₆₉ is P. Subgenus 5.7: X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉(SEQ ID NO: 359), wherein X₆₂ is A, G, I, M, P, S, T, or V; X₆₃ is L, Q,S, or V; X₆₄ is A, C, F, K, L, Q, R or S; X₆₅ is D, G, S, or V; X₆₆ is Lor M; X₆₇ is G, I, L, M, N, Q, or R; X₆₈ is I, N, P, or S; and X₆₉ is A,H, I, N, Q, or S. Subgenus 5.8: X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ (SEQ ID NO:360), wherein X₆₂ is A, I, or S; X₆₃ is L, Q, S, or V; X₆₄ is L, R or S;X₆₅ is G; X₆₆ is L; X₆₇ is L, M, or R; X₆₈ is S; and X₆₉ is A, H, N, Q,or S. Subgenus 5.9: X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ (SEQ ID NO: 361), whereinX₆₂ is A, I, or S; X₆₃ is L, Q, S, or V; X₆₄ is L, R or S; X₆₅ is G; X₆₆is L; X₆₇ is L, M, or R; X₆₈ is S; and X₆₉ is A, H, N, Q, or S. Subgenus5.10: X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ (SEQ ID NO: 362), wherein X₆₂ is A or S;X₆₃ is L or V; X₆₄ is L or S; X₆₅ is G; X₆₆ is L; X₆₇ is L or R; X₆₈ isS; and X₆₉ is H, or S. Subgenus 5.11: X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉ (SEQ IDNO: 363), wherein X₆₂ is A or S; X₆₃ is L or V; X₆₄ is S; X₆₅ is G; X₆₆is L; X₆₇ is L or R; X₆₈ is S; and X₆₉ is H, or S.

TABLE 8F-1 MMP14 Cleavable Core CM Consensus Sequence 6 Core CMConsensus 6 Subgenus of Core CM Consensus 6 X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉Subgenus 6.1: X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉ (SEQ ID NO: 372), (SEQ ID NO:371), wherein wherein X₇₂ is A, F, G, H, I, L, M, Q, R, or S; X₇₃ is A,F, H, L, X₇₂ is A, C, D, E, F, G, H, or N; X₇₄ is A, E, N, Q, or S; X₇₅is A, E, K, N, S, or T; X₇₆ is L I, K, L, M, N, P, Q, R, S, or M; X₇₇ isA, I, K, L, P, R, or V; X₇₈ is A, D, I, L, M, R, T, or or V; V; and X₇₉is A, F, G, H, I, L, P, Q, R, or S. X₇₃ is A, C, E, F, H, L, N, Subgenus6.2: X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉ (SEQ ID NO: 373), R, S, or V; wherein X₇₂is G, L or R, or S; X₇₃ is A or L; X₇₄ is A, E, N, Q, X₇₄ is A, D, E, K,N, P Q, or S; X₇₅ is A, E, N, S, or T; X₇₆ is L or M; X₇₇ is L or R; X₇₈is S, T, or Y; A, L, or T; and X₇₉ is F, G, L, R, or S. X₇₅ is A, E, G,H, K, L, N, Subgenus 6.3: X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉ (SEQ ID NO: 374), P,R, S, or T; wherein X₇₂ is L; X₇₃ is A or L; X₇₄ is E, N, Q, or S; X₇₅is A or X₇₆ is I, K, L, M, N, R, T, S; X₇₆ is L or M; X₇₇ is R; X₇₈ is Aor T; and X₇₉ is F, L, or R. V or Y; Subgenus 6.4:X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉ (SEQ ID NO: 375), X₇₇ is A, D, E, I, K, L, P,wherein X₇₂ is L; X₇₃ is A or L; X₇₄ is E, N, Q, or S; X₇₅ is A or Q, R,S, T, V, or Y; S; X₇₆ is L or M; X₇₇ is R; X₇₈ is A; and X₇₉ is L or R.X₇₈ is A, C, D, E, G, I, L, M, Q, R, S, T, or V; and X₇₉ is A, F, G, H,I, L, P, Q, R, S, T, or Y.

TABLE 8F-2 MMP14 Cleavable Core CM Consensus Sequence 6A Core CMConsensus 6A Subgenus of Core CM Consensus 6A X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈ (SEQID Subgenus 6A.1: X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈ (SEQ ID NO: 485), wherein NO:376), wherein X₇₂ is A, E, L, N, P, or Q; X₇₃ is X₇₂ is A, C, D, E, F,G, H, I, K, L, M, F, H, L, N, or S; X₇₄ is Q or Y; X₇₅ is A; X₇₆ is L,T, N, P, Q, R, S, or V; V or Y; X₇₇ is D, E, P, Q, or R; and X₇₈ is A,C, G, I, X₇₃ is A, C, E, F, H, L, N, R, S, or V; M, R, S, or T. X₇₄ isA, D, E, K, N, P Q, S, T, or Y; Subgenus 6A.2: X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈(SEQ ID X₇₅ is A, E, G, H, K, L, N, P, R, S, or NO: 377), wherein X₇₂ isA, E, L, or Q; X₇₃ is F, H, T; or N; X₇₄ is Q; X₇₅ is A; X₇₆ is L or T;X₇₇ is Q or R; X₇₆ is I, K, L, M, N, R, T, V or Y; and X₇₈ is I or M.X₇₇ is A, D, E, I, K, L, P, Q, R, S, T, Subgenus 6A.3:X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈ (SEQ ID V, or Y; and NO: 378), wherein X₇₂ is A;X₇₃ is F, H, or N; X₇₄ is X₇₈ is A, C, D, E, G, I, L, M, Q, R, S, Q; X₇₅is A; X₇₆ is L; X₇₇ is R; and X₇₈ is M. T, or V.

TABLE 8G MMP14 Cleavable Core CM Consensus Sequence 7 Core CM Consensus7 Subgenus of Core CM Consensus 7 X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉ Subgenus 7.1:X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉ (SEQ ID NO: 395), (SEQ ID NO: 394), whereinwherein X₈₂ is L; X₈₃ is H, K, Q, R, or Y; X₈₄ is A, L, M, S, T, X₈₂ isA, F, L, Q, S, T, or V; or V; X₈₅ is A, I, L, S, or V; X₈₆ is P; X₈₇ isA, G, R, S, V, or X₈₃ is A, E, G, H, K, Q, R, V, W; X₈₈ is I, R, T, orW; and X₈₉ is A, F, G, L, S, or V. or Y; Subgenus 7.2:X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉ (SEQ ID NO: 396), X₈₄ is A, G, I, K, L, M, N,S, wherein X₈₂ is L; X₈₃ is H, K, R, or Y; X₈₄ is A, L, or V; X₈₅ is T,or V; A, I, or L; X₈₆ is P; X₈₇ is G, R, or V; X₈₈ is T or W; and X₈₉ isX₈₅ is A, D, F, G, I, L, N, P, A, F, L, or S. R, S, T, or V; Subgenus7.3: X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉ (SEQ ID NO: 397), X₈₆ is A, P, or R;wherein X₈₂ is L; X₈₃ is K, R, or Y; X₈₄ is A; X₈₅ is A or L; X₈₇ is A,D, G, L, M, P, R, S, X₈₆ is P; X₈₇ is G, R, or V; X₈₈ is W; and X₈₉ is Aor L. T, V, W, or Y; Subgenus 7.4: X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉ (SEQ ID NO:398), X₈₈ is A, C, E, F, H, I, L, N, wherein X₈₂ is A, F, L, Q, or S;X₈₃ is A, E, G, H, K, Q, or Y; R, S, T, W, or Y; and X₈₄ is A, G, K, S,or V; X₈₅ is A, I, L, P, or T; X₈₆ is A, P, or X₈₉ is A, F, G, I, L, M,R, S, T, or R; X₈₇ is A, L, M, R, V, or Y; X₈₈ is C, H, R, T, or W; andX₈₉ V. is A, F, L, R, S, or T. Subgenus 7.5: X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉(SEQ ID NO: 399), wherein X₈₂ is F or L; X₈₃ is G, K, Q, or Y; X₈₄ is A,G, S, or V; X₈₅ is A, I, or L; X₈₆ is P; X₈₇ is A, R, or V; X₈₈ is R orW; and X₈₉ is A, F, L, or R. Subgenus 7.6: X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉ (SEQID NO: 400), wherein X₈₂ is L; X₈₃ is K or Y; X₈₄ is A or S; X₈₅ is A,I, or L; X₈₆ is P; X₈₇ is A, R, or V; X₈₈ is W; and X₈₉ is A or F.Subgenus 7.7: X₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉ (SEQ ID NO: 401), wherein X₈₂ isL; X₈₃ is K or Y; X₈₄ is A; X₈₅ is A or I; X₈₆ is P; X₈₇ is R or V; X₈₈is W; and X₈₉ is A or F.

TABLE 8H-1 MMP14 Cleavable Core CM Consensus Sequence 8 Core CMConsensus 8 Subgenus of Core CM Consensus 8 X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈Subgenus 8.1: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈ (SEQ ID NO: 411), wherein (SEQ IDNO: 410), X₉₂ is A, F, G, I, L, M, N, S, T, V, or W; X₉₃ is P; X₉₄ is A,E, F, H, wherein I, K, N, P, Q, R, S, T, or V; X₉₅ is A, D, E, G, H, N,P, or S; X₉₆ is X₉₂ is A, D, F, G, H, I, C, F, I, L, M, R, S, or V; X₉₇is C, F, G, I, L, R, S, T, V, or Y; and L, M, N, P, Q, R, S, X₉₈ is A,F, L, M, P, Q, R, S, T, V, or Y. T, V, or W; Subgenus 8.2:X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈ (SEQ ID NO: 412), wherein X₉₃ is A, P, R, or T;X₉₂ is F, G, L, S, T, or V; X₉₃ is P; X₉₄ is A, E, H, K, N, Q, R, S, T,X₉₄ is A, E, F, G, H, I, or V; X₉₅ is A, G, H, N, P, or S; X₉₆ is I, L,M, or V; X₉₇ is F, I, L, K, L, N, P, Q, R, S, T, R, S, T, V, or Y; andX₉₈ is A, F, L, R, T, V, or Y. or V; Subgenus 8.3: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈(SEQ ID NO: 413), wherein X₉₅ is A, D, E, G, H, K, X₉₂ is F, L, or S;X₉₃ is P; X₉₄ is A, K, Q, R, or S; X₉₅ is A, G, H, or M, N, P, R, S, orT; S; X₉₆ is I, L, M, or V; X₉₇ is F, L, R, S, T, V, or Y and; X₉₈ is F,L, X₉₆ is C, F, H, I, L, M, T, or V. P, R, S, V, W, or Y; Subgenus 8.4:X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈ (SEQ ID NO: 414), wherein X₉₇ is A, C, F, G, H, I,X₉₂ is F, L, or S; X₉₃ is P; X₉₄ is A, Q, or S; X₉₅ is G or S; X₉₆ is I,K, L, M, R, S, T, V, L, or M; X₉₇ is L, S, or V; and X₉₈ is F, L, or T.W, or Y; and Subgenus 8.5: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈ (SEQ ID NO: 415),wherein X₉₈ is A, D, E, F, G, H, X₉₂ is F, L, or S; X₉₃ is P; X₉₄ is A,Q or S; X₉₅ is G; X₉₆ is I, L, or I, K, L, M, N, P, Q, R, S, T, M; X₉₇is L or V; and X₉₈ is L. V, or Y. Subgenus 8.6: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈(SEQ ID NO: 416), wherein X₉₂ is F, L, or S; X₉₃ is P; X₉₄ is A or S;X₉₅ is G; X₉₆ is I, L, or M; X₉₇ is L or V; and X₉₈ is L. Subgenus 8.7:X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈ (SEQ ID NO: 417), wherein X₉₂ is F, G, L, M, P, S,V, or W; X₉₃ is P; X₉₄ is A, N, Q, or S; X₉₅ is A, D, G, H, M, N, P, orS; X₉₆ is F, I, L, M, or V; X₉₇ is A, I, L, M, S, or V; and X₉₈ is A, G,I, L, M, N, P, Q, R, S, T, or Y. Subgenus 8.8: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈(SEQ ID NO: 418), wherein X₉₂ is L, S, or V; X₉₃ is P; X₉₄ is A, N, Q,or S; X₉₅ is H, N, P, or S; X₉₆ is F, I, L, or M; X₉₇ is I, L, S, or V;and X₉₈ is A, L, or Q. Subgenus 8.9: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈ (SEQ ID NO:419), wherein X₉₂ is L; X₉₃ is P; X₉₄ is A, N, Q, or S; X₉₅ is H; X₉₆ isI or L; X₉₇ is V; and X₉₈ is L.

TABLE 8H-2 MMP14 Cleavable Extended Core CM Consensus Sequence 8Extended Core CM Subgenus of Extended Core CM Consensus 8A Consensus 8AX₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈X₉₉ Subgenus 8A.1: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈X₉₉ (SEQ IDNO: 487), (SEQ ID NO: 486), wherein X₉₂ is A, F, G, I, L, M, N, S, T, V,or W; X₉₃ is P; X₉₄ is wherein A, E, F, H, I, K, N, P, Q, R, S, T, or V;X₉₅ is A, D, E, G, H, N, P, X₉₂ is A, D, F, G, H, I, L, or S; X₉₆ is C,F, I, L, M, R, S, or V; X₉₇ is C, F, G, I, L, R, S, T, M, N, P, Q, R, S,T, V, V, or Y; X₉₈ is A, F, L, M, P, Q, R, S, T, V, or Y; and X₉₉ is A,D, or W; E, G, H, I, L, N, P, Q, R, S, T, V, W, or Y. X₉₃ is A, P, R, orT; Subgenus 8A.2: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈X₉₉ (SEQ ID NO: 488), X₉₄ is A,E, F, G, H, I, K, wherein X₉₂ is F, G, L, S, T, or V; X₉₃ is P; X₉₄ isA, E, H, K, N, L, N, P, Q, R, S, T, or Q, R, S, T, or V; X₉₅ is A, G, H,N, P, or S; X₉₆ is I, L, M, or V; V; X₉₇ is F, I, L, R, S, T, V, or Y;X₉₈ is A, F, L, R, T, V, or Y; and X₉₅ is A, D, E, G, H, K, X₉₉ is A, D,G, L, P, R, S, T, V, or Y. M, N, P, R, S, or T; Subgenus 8A.3:X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈X₉₉ (SEQ ID NO: 489), X₉₆ is C, F, H, I, L, M, P,wherein X₉₂ is F, L, or S; X₉₃ is P; X₉₄ is A, K, Q, R, or S; X₉₅ is R,S, V, W, or Y; A, G, H, or S; X₉₆ is I, L, M, or V; X₉₇ is F, L, R, S,T, V, or Y; X₉₇ is A, C, F, G, H, I, K, X₉₈ is F, L, T, or V; and X₉₉ isA, D, G, L, R, T, or V. L, M, R, S, T, V, W, or Subgenus 8A.4:X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈X₉₉ (SEQ ID NO: 490), Y; wherein X₉₂ is F, L, or S;X₉₃ is P; X₉₄ is A, Q, or S; X₉₅ is G or S; X₉₈ is A, D, E, F, G, H, I,K, X₉₆ is I, L, or M; X₉₇ is L, S, or V; X₉₈ is F, L, or T; and X₉₉ isA, L, M, N, P, Q, R, S, R, or T. T, V, or Y; and Subgenus 8A.5:X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈X₉₉ (SEQ ID NO: 491), X₉₉ is A, D, E, F, G, H, I,wherein X₉₂ is F, L, or S; X₉₃ is P; X₉₄ is A, Q or S; X₉₅ is G; X₉₆ K,L, N, P, Q, R, S, T, V, W, is I, L, or M; X₉₇ is L or V; X₉₈ is L; andX₉₉ is R. or Y. Subgenus 8A.6: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈X₉₉ (SEQ ID NO:492), wherein X₉₂ is F, L, or S; X₉₃ is P; X₉₄ is A or S; X₉₅ is G; X₉₆is I, L, or M; X₉₇ is L or V; X₉₈ is L; and X₉₉ is R. Subgenus 8A.7:X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈X₉₉ (SEQ ID NO: 493), wherein X₉₂ is F, G, L, M, P,S, V, or W; X₉₃ is P; X₉₄ is A, N, Q, or S; X₉₅ is A, D, G, H, M, N, P,or S; X₉₆ is F, I, L, M, or V; X₉₇ is A, I, L, M, S, or V; X₉₈ is A, G,I, L, M, N, P, Q, R, S, T, or Y; and X₉₉ is A, F, H, I, L, Q, R, T, V,W, or Y. Subgenus 8A.8: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈X₉₉ (SEQ ID NO: 494),wherein X₉₂ is L, S, or V; X₉₃ is P; X₉₄ is A, N, Q, or S; X₉₅ is H, N,P, or S; X₉₆ is F, I, L, or M; X₉₇ is I, L, S, or V; X₉₈ is A, L, or Q;and X₉₉ is L, T, V, or Y. Subgenus 8A.9: X₉₂X₉₃X₉₄X₉₅X₉₆X₉₇X₉₈X₉₉ (SEQID NO: 495), wherein X₉₂ is L; X₉₃ is P; X₉₄ is A, N, Q, or S; X₉₅ is H;X₉₆ is I or L; X₉₇ is V; X₉₈ is L; and X₉₉ is L or V.

TABLE 8I MMP14 Cleavable Core CM Consensus Sequence 9 Core CM Consensus9 Subgenus of Core CM Consensus 9 X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉Subgenus 9.1: X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉ (SEQ ID (SEQ ID NO: 425),wherein NO: 426), wherein X₁₀₂ is A, D, F, G, I, R, or S; X₁₀₃ is D,X₁₀₂ is A, D, F, G, H, I, L, M, P, E, L, M, P, R, S, T, V, or Y; X₁₀₄ isA, H, P, or S; X₁₀₅ is R, S, T, or V; A, D, E, H, L, M, N, R, T, or V;X₁₀₆ is A, G, or R; X₁₀₇ is X₁₀₃ is A, D, E, L, M, P, Q, R, S, F, L, M,S, V, or W; X₁₀₈ is A, E, H, L, M, R, S, or V; and T, V, or Y; X₁₀₉ isA, G, L, P, R, S, or V. X₁₀₄ is A, G, H, L, N, P, R, S, T, Subgenus 9.2:X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉ (SEQ ID or V; NO: 427), wherein X₁₀₂ isF, G, I, R, or S; X₁₀₃ is L, P, R, X₁₀₅ is A, D, E, H, L, M, N, P, Q, orV; X₁₀₄ is A or H; X₁₀₅ is A, D, or R; X₁₀₆ is A or G; R, S, T, or V;X₁₀₇ is L or V; X₁₀₈ is H, L, M, R, S, or V; and X₁₀₉ is A, X₁₀₆ is A,G, R, S, or T; L, S, or V. X₁₀₇ is C, F, L, M, S, V, W, or Y; Subgenus9.3: X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉ (SEQ ID X₁₀₈ is A, E, F, G, H, I,L, M, N, NO: 428), wherein X₁₀₂ is G, R or S; X₁₀₃ is R or V; X₁₀₄ is Q,R, S, V, W, or Y; and A or H; X₁₀₅ is A, D, or R; X₁₀₆ is A or G; X₁₀₇is L or V; X₁₀₉ is A, E, G, L, P, R, S, or V. X₁₀₈ is H or R; and X₁₀₉is A, L, S, or V. Subgenus 9.4: X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉ (SEQ IDNO: 429), wherein X₁₀₂ is R; X₁₀₃ is R; X₁₀₄ is A or H; X₁₀₅ is A or D;X₁₀₆ is G; X₁₀₇ is L or V; X₁₀₈ is R; and X₁₀₉ is A, S, or V. Subgenus9.5: X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉ (SEQ ID NO: 430), wherein X₁₀₂ isD, F, G, I, L, R, S, or T; X₁₀₃ is E, L, M, R, S, T, or V; X₁₀₄ is H orN; X₁₀₅ is A, D, L, M, R, or T; X₁₀₆ is A, G, R, or T; X₁₀₇ is C, L, M,S, V, or W; X₁₀₈ is A, E, F, G, L, R, S, or W; and X₁₀₉ is A, G, L, P,R, S, or V. Subgenus 9.6: X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉ (SEQ ID NO:431), wherein X₁₀₂ is F, I, R, or S; X₁₀₃ is E, L, R, or V; X₁₀₄ is H;X₁₀₅ is D, M, R, or T; X₁₀₆ is A or G; X₁₀₇ is L, M, S, or V; X₁₀₈ is E,R, or S; and X₁₀₉ is A, P, S, or V. Subgenus 9.7:X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉ (SEQ ID NO: 432), wherein X₁₀₂ is I orR; X₁₀₃ is E, R, or V; X₁₀₄ is H; X₁₀₅ is D, M, R, or T; X₁₀₆ is A or G;X₁₀₇ is L or V; X₁₀₈ is R or S; and X₁₀₉ is A, P, S, or V. Subgenus 9.8:X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉ (SEQ ID NO: 433), wherein X₁₀₂ is I orR; X₁₀₃ is R; X₁₀₄ is H; X₁₀₅ is D; X₁₀₆ is A or G; X₁₀₇ is L or V; X₁₀₈is R or S; and X₁₀₉ is A or S.

TABLE 8J MMP14 Cleavable Core CM Consensus Sequence 10 Core CM Consensus10 Subgenus of Core CM Consensus 10 X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉Subgenus 10.1: X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ (SEQ ID NO: 436),wherein ID NO: 437), wherein X₁₁₂ is A, I, P, S, T, V, or Y; X₁₁₃ isX₁₁₂ is A, D, G, H, I, L, N, P, R, A, D, G, L, M, Q, R, S, V, or Y; X₁₁₄is A, H, K, L, N, S, S, T, V, W, or Y; or T; X₁₁₅ is G, H, I, L, S, orV; X₁₁₆ is I, L, or V; X₁₁₇ is X₁₁₃ is A, D, G, H, L, M, N, P, A, F, G,K, R, S, or W; X₁₁₈ is D, H, L, M, N, Q, R, or V; Q, R, S, V, or Y; andX₁₁₉ is A, I, L, or V. X₁₁₄ is A, H, K, L, N, P, Q, R, S, Subgenus 10.2:X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ T, or V; ID NO: 438), wherein X₁₁₂is A, I, T, or V; X₁₁₃ is A, L, M, X₁₁₅ is A, D, F, G, H, I, L, P, R, Q,R, V, or Y; X₁₁₄ is A, N, S, or T; X₁₁₅ is G, L, S, or V; S, V, or Y;X₁₁₆ is L or V; X₁₁₇ is A, F, G, K, or S; X₁₁₈ is M, N, Q, R, X₁₁₆ is C,F, I, L, P, V, or Y; or V; and X₁₁₉ is I, L, or V. X₁₁₇ is A, D, E, F,G, I, K, M, N, Subgenus 10.3: X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ R,S, T, V, or W; ID NO: 439), wherein X₁₁₂ is A, I, T, or V; X₁₁₃ is M, Q,or X₁₁₈ is A, D, E, F, H, K, L, M, N, Y; X₁₁₄ is A, N, or S; X₁₁₅ is G,L, S, or V; X₁₁₆ is L or V; Q, R, V, or Y; and X₁₁₇ is A, F, G, or S;X₁₁₈ is M, N, Q, or R; and X₁₁₉ is I, L, X₁₁₉ is A, F, I, L, M, or V. orV. Subgenus 10.4: X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ ID NO: 440),wherein X₁₁₂ is A, I, or V; X₁₁₃ is Y; X₁₁₄ is N or S; X₁₁₅ is G, L, orV; X₁₁₆ is L; X₁₁₇ is A, G, or S; X₁₁₈ is M, Q, or R; and X₁₁₉ is L orV. Subgenus 10.5: X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ ID NO: 441),wherein X₁₁₂ is A, I, or V; X₁₁₃ is Y; X₁₁₄ is N or S; X₁₁₅ is G, L, orV; X₁₁₆ is L; X₁₁₇ is G or S; X₁₁₈ is M or R; and X₁₁₉ is L or V.Subgenus 10.6: X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ ID NO: 442),wherein X₁₁₂ is A, I, or V; X₁₁₃ is Y; X₁₁₄ is N or S; X₁₁₅ is G, L, orV; X₁₁₆ is L; X₁₁₇ is S, X₁₁₈ is M or R; and X₁₁₉ is L or V. Subgenus10.7: X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ ID NO: 443), wherein X₁₁₂ isA; X₁₁₃ is Y; X₁₁₄ is N or S; X₁₁₅ is G or L; X₁₁₆ is L; X₁₁₇ is S; X₁₁₈is R; and X₁₁₉ is L or V. Subgenus 10.8:X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ ID NO: 444), wherein X₁₁₂ is A, D,G, I, L, N, P, S, T, V, W, or Y; X₁₁₃ is A, D, G, L, M, Q, S, or V; X₁₁₄is H, K, N, P, Q, R, S, or T; X₁₁₅ is H, I, L, R, or V; X₁₁₆ is I, L, P,or V; X₁₁₇ is A, D, E, G, I, K, M, N, S, or T; X₁₁₈ is D, F, L, M, Q, R,or V; and X₁₁₉ is A, F, I, L, or V. Subgenus 10.9:X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ ID NO: 445), wherein X₁₁₂ is A, I,T, or V; X₁₁₃ is A, D, G, L, M, Q, S, or V; X₁₁₄ is H, K, N, S, or T;X₁₁₅ is H, I, L, or V; X₁₁₆ is L; X₁₁₇ is A, G, K, or S; X₁₁₈ is L, M,Q, R, or V; and X₁₁₉ is A, L, or V. Subgenus 10.10:X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ ID NO: 446), wherein X₁₁₂ is A orI; X₁₁₃ is A, L, or Q; X₁₁₄ is N, S, or T; X₁₁₅ is L or V; X₁₁₆ is L;X₁₁₇ is A, G, K, or S; X₁₁₈ is M, R, or V; and X₁₁₉ is L or V. Subgenus10.11: X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ ID NO: 447), wherein X₁₁₂is A or I; X₁₁₃ is A, L, or Q; X₁₁₄ is N or S; X₁₁₅ is L or V; X₁₁₆ isL; X₁₁₇ is A or S; X₁₁₈ is M or R; and X₁₁₉ is L or V. Subgenus 10.12:X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ ID NO: 448), wherein X₁₁₂ is I;X₁₁₃ is A, L, or Q; X₁₁₄ is N; X₁₁₅ is L or V; X₁₁₆ is L; X₁₁₇ is A orS; X₁₁₈ is M or R; and X₁₁₉ is L or V. Subgenus 10.13:X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉ (SEQ ID NO: 449), wherein X₁₁₂ is I;X₁₁₃ is A, L, or Q; X₁₁₄ is N; X₁₁₅ is L or V; X₁₁₆ is L; X₁₁₇ is S,X₁₁₈ is M; and X₁₁₉ is L or V.

TABLE 8K MMP14 Cleavable Core CM Consensus Sequence 11 Core CM Consensus11 Subgenus of Core CM Consensus 11 X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉Subgenus 11.1: X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉ (SEQ ID (SEQ ID NO:453), wherein NO: 454), wherein X₁₂₂ is A, G, P, R, or S; X₁₂₃ is A, R,or X₁₂₂ is A, G, H, L, P, R, S, or V; S; X₁₂₄ is G, P, S, or T; X₁₂₅ isL or V; X₁₂₆ is W; X₁₂₇ is L, X₁₂₃ is A, G, R, S, T or V; S, V, or Y;X₁₂₈ is D, E, P, or T; and X₁₂₉ is P, Q or V. X₁₂₄ is A, G, P, R, S, orT; Subgenus 11.2: X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉ (SEQ ID X₁₂₅ is H, I,L, P, R, or V; NO: 455), wherein X₁₂₂ is G, P, R, or S; X₁₂₃ is A or R;X₁₂₄ X₁₂₆ is L or W; is G, P, or S; X₁₂₅ is L or V; X₁₂₆ is W; X₁₂₇ is Lor Y; X₁₂₈ X₁₂₇ is F, H, L, M, Q, S, V, or Y; is E or T; and X₁₂₉ is Q.X₁₂₈ is A, D, E, I, K, P, R, S, T, or Subgenus 11.3:X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉ (SEQ ID V; and NO: 456), wherein X₁₂₂is P; X₁₂₃ is A; X₁₂₄ is P or S; X₁₂₅ X₁₂₉ is A, E, F, G, H, I, L, N, P,Q, is L or V; X₁₂₆ is W; X₁₂₇ is Y; X₁₂₈ is T; and X₁₂₉ is Q. R, or V.

TABLE 8L MMP14 Cleavable Core CM Consensus Sequence 12 Core CM Consensus12 Subgenus of Core CM Consensus 12 X₂X₃X₄X₅X₆X₇X₈X₉ Subgenus 12.1:X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO: 459), wherein (SEQ ID NO: 458), X₂ is A, G,L, P, or S; X₃ is A, E, G, H, L, P, Q, S, T, or V; X₄ is G, wherein N,R, or S; X₅ is L, P, or S; X₆ is I or L; X₇ is A, G, N, Q, R, or S; X₂is A, D, E, F, G, H, X₈ is D, F, G, I, L, M, P, S, or V; and X₉ is F, G,L, P, Q, R, or S. I, L, M, N, P, Q, R, S, Subgenus 12.2:X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO: 460), wherein T, V, or Y; X₂ is A, P, or S;X₃ is L, S or V; X₄ is G, N, R, or S; X₅ is L, P, or S; X₃ is A, E, F,G, H, I, X₆ is L; X₇ is A, G, R, or S; X₈ is L, P, or V; and X₉ is F, L,P, or S. K, L, M, P, Q, R, S, Subgenus 12.3: X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ IDNO: 461), wherein T, V, W, or Y; X₂ is A, P, or S; X₃ is L, S, or V; X₄is G, N, R, or S; X₅ is L, P, or X₄ is A, E, G, H, K, N, S; X₆ is L; X₇is A, G, R, or S; X₈ is L or P; and X₉ is F, P, or S. P, R, S, T, V, orY; Subgenus 12.4: X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO: 462), wherein X₅ is A, G,H, I, L, N, X₂ is A, P, or S; X₃ is L or V; X₄ is G, N, or S; X₅ is L orS; X₆ is L; P, R, S, T, or V; X₇ is A, G, R, or S; X₈ is L or P; and X₉is P or S. X₆ is I, L, M, Q, T, V, W, or Subgenus 12.5: X₂X₃X₄X₅X₆X₇X₈X₉(SEQ ID NO: 463), wherein Y; X₂ is A or S; X₃ is L; X₄ is G, N, or S; X₅is L or S; X₆ is L; X₇ is R X₇ is A, D, G, H, K, L, or S; X₈ is L; andX₉ is P. N, P, Q, R, S, T, or V; Subgenus 12.6: X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ IDNO: 464), wherein X₈ is A, D, E, F, G, I, X₂ is A, E, G, H, I, L, M, P,or S; X₃ is A, E, G, H, I, K, L, P, Q, R, K, L, M, P, Q, R, S, S, T, V,W, or Y; X₄ is A, G, N, R, S, T, or V; X₅ is A, G, H, L, N, V, W, or Y;and P, R, S, T, or V; X₆ is I, L, M, or Q; X₇ is A, D, G, K, L, N, Q, R,S, X₉ is A, F, G, I, L, M, or V; X₈ is A, D, E, F, G, I, K, L, M, P, R,V, W, or Y; and X₉ is A, N, P, Q, R, S, T, V or Y. F, G, M, P, Q, R, S,V, or Y. Subgenus 12.7: X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO: 465), wherein X₂ isA, P, or S; X₃ is A, H, Q, S, or V; X₄ is G, N, or S; X₅ is L, P, or S;X₆ is L; X₇ is A, D, G, R, or S; X₈ is F, I, L, M, or P; and X₉ is F, P,Q, or R. Subgenus 12.8: X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO: 466), wherein X₂ isA, P, or S; X₃ is H, S, or V; X₄ is G, N, or S; X₅ is L, P, or S; X₆ isL; X₇ is A, G, R, or S; X₈ is F, I, M, or P; and X₉ is P or R. Subgenus12.9: X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO: 467), wherein X₂ is A, P, or S; X₃ isS or V; X₄ is G, N, or S; X₅ is L; X₆ is L; X₇ is A, G or R; X₈ is F, I,or P; and X₉ is P. Subgenus 12.10: X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO: 468),wherein X₂ is A, P, or S; X₃ is S or V; X₄ is G, N, or S; X₅ is L; X₆ isL; X₇ is A or R; X₈ is F or P; and X₉ is P. Subgenus 12.11:X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO: 469), wherein X₂ is A or P; X₃ is S; X₄ isG or N; X₅ is L; X₆ is L; X₇ is R; X₈ is F; and X₉ is P.

TABLE 8M MMP14 Cleavable Core CM Consensus Sequence 13 Core CM Consensus13 Subgenus of Core CM Consensus 13 X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉ Subgenus13.1: X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉ (SEQ ID NO: 475), (SEQ ID NO: 473),wherein wherein X₁₂ is F, I, L, M, S, or V; X₁₃ is A, E, H, K, L, M, N,X₁₂ is F, I, L, M, R, S, T, or Q, S, T, V, or Y; X₁₄ is A, F, H, L, M,Q, S, T, or V; X₁₅ is A, V; G, or P; X₁₆ is A, F, G, H, I, L, M, N, R,S, V, or Y; X₁₇ is A, E, X₁₃ is A, E, G, H, I, K, L, G, H, L, M, P, Q,R, S, T, or V; X₁₈ is A, D, E, F, G, H, L, M, M, N, P, Q, R, S, T, V, W,or Y; N, R, S, V, or Y; and X₁₉ is A, F, G, I, L, M, P, Q, R, S, W, orY. X₁₄ is A, D, E, F, G, H, I, K, Subgenus 13.2:X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉ (SEQ ID NO: 476), L, M, N, P, Q, R, S, T, V,wherein X₁₂ is L, M, or V; X₁₃ is A, H, L, N, Q, S, or V; X₁₄ is or Y;A, L, M, Q, S, T, or V; X₁₅ is P; X₁₆ is A, F, G, I, L, R, S, V, or X₁₅is A, E, G, N, P, Q, S, T, Y; X₁₇ is H, L, M, Q, or S; X₁₈ is A, D, G,H, R, or S; and X₁₉ is V, or W; A, F, G, L, R, or S. X₁₆ is A, F, G, H,I, K, L, Subgenus 13.3: X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉ (SEQ ID NO: 477), M, N,P, Q, R, S, T, V, or wherein X₁₂ is L, M, or V; X₁₃ is A or L; X₁₄ is A,L, or S; X₁₅ Y; is P; X₁₆ is L or V; X₁₇ is H, L, or Q; X₁₈ is G or S;and X₁₉ is X₁₇ is A, D, E, F, G, H, I, L, G, R, or S. M, N, P, Q, R, S,T, V, or Subgenus 13.4: X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉ (SEQ ID NO: 478), Y;wherein X₁₂ is L or V; X₁₃ is A or L; X₁₄ is L or S; X₁₅ is P; X₁₆ X₁₈is A, C, D, E, F, G, H, I, is L or V; X₁₇ is H or L; X₁₈ is G or S; andX₁₉ is R or S. L, M, N, P, Q, R, S, T, V, or Y; and Subgenus 13.5:X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉ (SEQ ID NO: 479), X₁₉ is A, D, F, G, H, I, L,wherein X₁₂ is L or V; X₁₃ is A or L; X₁₄ is L or S; X₁₅ is P; X₁₆ M, N,P, Q, R, S, T, V, W, or Y. is L; X₁₇ is H or L; X₁₈ is G; and X₁₉ is S.Subgenus 13.6: X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉ (SEQ ID NO: 480), wherein X₁₂ isF, I, L, M, S, T, or V; X₁₃ is A, E, G, H, L, M, S, V, W, or Y; X₁₄ isA, D, E, G, K, L, M, N, Q, R, S, T, or V; X₁₅ is E, G, N, P, S, T, or V;X₁₆ is A, F, G, L, N, P, Q, R, S, V, or Y; X₁₇ is A, E, H, P, Q, or R;X₁₈ is D, E, G, N, R, S, or T; and X₁₉ is A, D, G, Q, S, T, or V.Subgenus 13.: X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉ (SEQ ID NO: 481), wherein X₁₂ isL, M, or V; X₁₃ is A or L; X₁₄ is A, L, Q, or S; X₁₅ is G, P, or T; X₁₆is A, S, or Y; X₁₇ is H or P; X₁₈ is D or G; and X₁₉ is A, G or S.Subgenus 13.7: X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉ (SEQ ID NO: 482), wherein X₁₂ isL or M; X₁₃ is A or L; X₁₄ is L; X₁₅ is G or P; X₁₆ is A or S; X₁₇ is H;X₁₈ is G; and X₁₉ is A or G.

In some embodiments, the CM comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 352, 371, 394, 410, 425, 436,453, 458, 473, 485, and 486. In some embodiments, the CM comprises anamino acid sequence selected from the group consisting of SEQ ID NO:353-363, 372-375, 376-378, 395-401, 411-419, 426-433, 437-449, 454-456,459-469, 475-482, and 487-495. In some embodiments, the CM comprises anamino acid sequence selected from the group consisting of SEQ ID NO:353-363. In some embodiments, the CM comprises an amino acid sequenceselected from the group consisting of 372-375. In some embodiments, theCM comprises an amino acid sequence selected from the group consistingof 376-378. In some embodiments, the CM comprises an amino acid sequenceselected from the group consisting of 395-401. In some embodiments, theCM comprises an amino acid sequence selected from the group consistingof 411-419. In some embodiments, the CM comprises an amino acid sequenceselected from the group consisting of 426-433. In some embodiments, theCM comprises an amino acid sequence selected from the group consistingof 437-449. In some embodiments, the CM comprises an amino acid sequenceselected from the group consisting of 454-456. In some embodiments, theCM comprises an amino acid sequence selected from the group consistingof 459-469. In some embodiments, the CM comprises an amino acid sequenceselected from the group consisting of 475-482. In some embodiments, theCM comprises an amino acid sequence selected from the group consistingof 487-495.

In some embodiments, the CM comprises an amino sequence selected fromthe group consisting of SEQ ID NOs: 317, 324, 329 and 340. In someembodiments, the CM comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 318-323, 325-327, 330-335, and 341-347.In some embodiments, the CM comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 318-323. In some embodiments,the CM comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 325-327. In some embodiments, the CM comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:330-335. In some embodiments, the CM comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 341-347.

In some embodiments, the CM comprises a core CM consensus 1 sequencecomprising the amino acid sequence RPSPMWAY (SEQ ID NO: 21).

In some embodiments, the CM comprises a core CM consensus 2 sequencecomprising the amino acid sequence WDHPISLL (SEQ ID NO: 328). In someembodiments, the CM comprises a core CM consensus 2 sequence comprisingthe amino acid sequence WATPRPMR (SEQ ID NO: 22).

In some embodiments, the CM comprises a core CM consensus 3 sequencecomprising the amino acid sequence LTFPTYIF (SEQ ID NO: 336). In someembodiments, the CM comprises a core CM consensus 3 sequence comprisingthe amino acid sequence MTFPTYIF (SEQ ID NO: 337). In some embodiments,the CM comprises a core CM consensus 3 sequence comprising the aminoacid sequence LTFPTYWF (SEQ ID NO: 338). In some embodiments, the CMcomprises a core CM consensus 3 sequence comprising the amino acidsequence MTFPTYWF (SEQ ID NO: 339). In some embodiments, the CMcomprises a core CM consensus 3 sequence comprising the amino acidsequence STFPFGMF (SEQ ID NO: 17).

In some embodiments, the CM comprises a core CM consensus 4 sequencecomprising the amino acid sequence DWLYWMGI (SEQ ID NO: 348). In someembodiments, the CM comprises a core CM consensus 4 sequence comprisingthe amino acid sequence DWLYWPGI (SEQ ID NO: 19). In some embodiments,the CM comprises a core CM consensus 4 sequence comprising the aminoacid sequence DWLYWMSI (SEQ ID NO: 349). In some embodiments, the CMcomprises a core CM consensus 4 sequence comprising the amino acidsequence DWLYWPSI (SEQ ID NO: 350). In some embodiments, the CMcomprises a core CM consensus 4 sequence comprising the amino acidsequence HWHLGPPT (SEQ ID NO: 351).

In some embodiments, the CM comprises a core CM consensus 5 sequencecomprising the amino acid sequence ISSGLLSS (SEQ ID NO: 14). In someembodiments, the CM comprises a core CM consensus 5 sequence comprisingthe amino acid sequence SVSGLLSH (SEQ ID NO: 364). In some embodiments,the CM comprises a core CM consensus 5 sequence comprising the aminoacid sequence SVSGLLSS (SEQ ID NO: 365). In some embodiments, the CMcomprises a core CM consensus 5 sequence comprising the amino acidsequence SVSGLRSH (SEQ ID NO: 366). In some embodiments, the CMcomprises a core CM consensus 5 sequence comprising the amino acidsequence SVSGLRSS (SEQ ID NO: 367). In some embodiments, the CMcomprises a core CM consensus 5 sequence comprising the amino acidsequence TLSGLRSP (SEQ ID NO: 368). In some embodiments, the CMcomprises a core CM consensus 5 sequence comprising the amino acidsequence TSSGLRSP (SEQ ID NO: 369). In some embodiments, the CMcomprises a core CM consensus 5 sequence comprising the amino acidsequence TVSGLRSP (SEQ ID NO: 370).

In some embodiments, the CM comprises a core CM consensus 6 sequencecomprising the amino acid sequence AFQALRM (SEQ ID NO: 379). In someembodiments, the CM comprises a core CM consensus 6 sequence comprisingthe amino acid sequence AHQALRM (SEQ ID NO: 380). In some embodiments,the CM comprises a core CM consensus 6 sequence comprising the aminoacid sequence ANQALRM (SEQ ID NO: 381). In some embodiments, the CMcomprises a core CM consensus 6 sequence comprising the amino acidsequence ANQALRMA (SEQ ID NO: 382). In some embodiments, the CMcomprises a core CM consensus 6 sequence comprising the amino acidsequence LLEALRAL (SEQ ID NO: 383). In some embodiments, the CMcomprises a core CM consensus 6 sequence comprising the amino acidsequence LLNALRAL (SEQ ID NO: 384). In some embodiments, the CMcomprises a core CM consensus 6 sequence comprising the amino acidsequence LLQALRAL (SEQ ID NO: 385). In some embodiments, the CMcomprises a core CM consensus 6 sequence comprising the amino acidsequence LLSALRAL (SEQ ID NO: 386). In some embodiments, the CMcomprises a core CM consensus 6 sequence comprising the amino acidsequence LLESLRAL (SEQ ID NO: 387). In some embodiments, the CMcomprises a core CM consensus 6 sequence comprising the amino acidsequence LLNSLRAL (SEQ ID NO: 388). In some embodiments, the CMcomprises a core CM consensus 6 sequence comprising the amino acidsequence LLQSLRAL (SEQ ID NO: 389). In some embodiments, the CMcomprises a core CM consensus 6 sequence comprising the amino acidsequence LLSSLRAL (SEQ ID NO: 390). In some embodiments, the CMcomprises a core CM consensus 6 sequence comprising the amino acidsequence QFQALRM (SEQ ID NO: 391). In some embodiments, the CM comprisesa core CM consensus 6 sequence comprising the amino acid sequenceQHQALRM (SEQ ID NO: 392). In some embodiments, the CM comprises a coreCM consensus 6 sequence comprising the amino acid sequence QNQALRM (SEQID NO: 393). In some embodiments, the CM comprises a core CM consensus 6sequence comprising the amino acid sequence QNQALRMA (SEQ ID NO: 15).

In some embodiments, the CM comprises a core CM consensus 7 sequencecomprising the amino acid sequence LKAAPRWA (SEQ ID NO: 24). In someembodiments, the CM comprises a core CM consensus 7 sequence comprisingthe amino acid sequence LKAAPVWA (SEQ ID NO: 403). In some embodiments,the CM comprises a core CM consensus 7 sequence comprising the aminoacid sequence LKAAPRWF (SEQ ID NO: 404). In some embodiments, the CMcomprises a core CM consensus 7 sequence comprising the amino acidsequence LKAAPVWF (SEQ ID NO: 405). In some embodiments, the CMcomprises a core CM consensus 7 sequence comprising the amino acidsequence LYAAPRWA (SEQ ID NO: 406). In some embodiments, the CMcomprises a core CM consensus 7 sequence comprising the amino acidsequence LYAAPVWA (SEQ ID NO: 407). In some embodiments, the CMcomprises a core CM consensus 7 sequence comprising the amino acidsequence LYAAPRWF (SEQ ID NO: 408). In some embodiments, the CMcomprises a core CM consensus 7 sequence comprising the amino acidsequence LYAAPVWF (SEQ ID NO: 409).

In some embodiments, the CM comprises a core CM consensus 8 sequencecomprising the amino acid sequence GPSHLVLT (SEQ ID NO: 25). In someembodiments, the CM comprises a core CM consensus 8 sequence comprisingthe amino acid sequence LPAGLLL (SEQ ID NO: 402). In some embodiments,the CM comprises a core CM consensus 8 sequence comprising the aminoacid sequence LPAGLLLR (SEQ ID NO: 420). In some embodiments, the CMcomprises a core CM consensus 8 sequence comprising the amino acidsequence LPAHLVLL (SEQ ID NO: 421). In some embodiments, the CMcomprises a core CM consensus 8 sequence comprising the amino acidsequence LPSHLVLL (SEQ ID NO: 422). In some embodiments, the CMcomprises a core CM consensus 8 sequence comprising the amino acidsequence LPAHLVLV (SEQ ID NO: 423). In some embodiments, the CMcomprises a core CM consensus 8 sequence comprising the amino acidsequence LPSHLVLV (SEQ ID NO: 424).

In some embodiments, the CM comprises a core CM consensus 9 sequencecomprising the amino acid sequence RMHLRSLG (SEQ ID NO: 29). In someembodiments, the CM comprises a core CM consensus 9 sequence comprisingthe amino acid sequence RRHDGLRA (SEQ ID NO: 434). In some embodiments,the CM comprises a core CM consensus 9 sequence comprising the aminoacid sequence RRHDGLRS (SEQ ID NO: 435).

In some embodiments, the CM comprises a core CM consensus 10 sequencecomprising the amino acid sequence AQNLLGMV (SEQ ID NO: 16). In someembodiments, the CM comprises a core CM consensus 10 sequence comprisingthe amino acid sequence IANLLSMV (SEQ ID NO: 450). In some embodiments,the CM comprises a core CM consensus 10 sequence comprising the aminoacid sequence ILNLLSMV (SEQ ID NO: 451). In some embodiments, the CMcomprises a core CM consensus 10 sequence comprising the amino acidsequence IQNLLSMV (SEQ ID NO: 452).

In some embodiments, the CM comprises a core CM consensus 11 sequencecomprising the amino acid sequence PAGLWLDP (SEQ ID NO: 33). In someembodiments, the CM comprises a core CM consensus 11 sequence comprisingthe amino acid sequence PASLWYTQ (SEQ ID NO: 457).

In some embodiments, the CM comprises a core CM consensus 12 sequencecomprising the amino acid sequence ALGLLRLP (SEQ ID NO: 470). In someembodiments, the CM comprises a core CM consensus 12 sequence comprisingthe amino acid sequence ALGLLSLP (SEQ ID NO: 471). In some embodiments,the CM comprises a core CM consensus 12 sequence comprising the aminoacid sequence ASGLLRFP (SEQ ID NO: 472). In some embodiments, the CMcomprises a core CM consensus 12 sequence comprising the amino acidsequence AVGLLAPP (SEQ ID NO: 31).

In some embodiments, the CM comprises a core CM consensus 13 sequencecomprising the amino acid sequence LAAPLGLL (SEQ ID NO: 30). In someembodiments, the CM comprises a core CM consensus 13 sequence comprisingthe amino acid sequence LLAPSHRA (SEQ ID NO: 32).

In some embodiments, the CM comprises a core CM consensus 13 sequencecomprising the amino acid sequence LLLPAHGG (SEQ ID NO: 474). In someembodiments, the CM comprises a core CM consensus 13 sequence comprisingthe amino acid sequence LLLPLLGS (SEQ ID NO: 483).

In some embodiments, the CM is a substrate for at least two proteases.In some embodiments, at least one protease is an MMP and at least oneprotease is selected from the group consisting of those shown in Table7.

TABLE 7 Exemplary Proteases and/or Enzymes ADAMS, ADAMTS, e.g. ADAM8ADAM9 ADAM10 ADAM12 ADAM15 ADAM17/TACE ADAMDEC1 ADAMTS1 ADAMTS4 ADAMTS5Aspartate proteases, e.g., BACE Renin Aspartic cathepsins, e.g.,Cathepsin D Cathepsin E Caspases, e.g., Caspase 1 Caspase 2 Caspase 3Caspase 4 Caspase 5 Caspase 6 Caspase 7 Caspase 8 Caspase 9 Caspase 10Caspase 14 Cysteine cathepsins, e.g., Cathepsin B Cathepsin C CathepsinK Cathepsin L Cathepsin S Cathepsin V/L2 Cathepsin X/Z/P Cysteineproteinases, e.g., Cruzipain Legumain Otubain-2 KLKs, e.g., KLK4 KLK5KLK6 KLK7 KLK8 KLK10 KLK11 KLK13 KLK14 Metallo proteinases, e.g., MeprinNeprilysin PSMA BMP-1 MMPs, e.g., MMP1 MMP2 MMP3 MMP7 MMP8 MMP9 MMP10MMP11 MMP12 MMP13 MMP14 MMP15 MMP16 MMP17 MMP19 MMP20 MMP23 MMP24 MMP26MMP27 Serine proteases, e.g., activated protein C Cathepsin A CathepsinG Chymase coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa,FXIIa) Elastase Granzyme B Guanidinobenzoatase HtrA1 Human NeutrophilElastase Lactoferrin Marapsin NS3/4A PACE4 Plasmin PSA tPA ThrombinTryptase uPA Type II Transmembrane Serine Proteases (TTSPs), e.g., DESC1DPP-4 FAP Hepsin Matriptase-2 MT-SP1/Matriptase TMPRSS2 TMPRSS3 TMPRSS4

In some embodiments, the antibody includes at least a first CM and asecond CM. In some embodiments, the first CM and the second CM are eachpolypeptides of no more than 15 amino acids long. In some embodiments,the first CM and the second CM in the antibody in the uncleaved statehave the structural arrangement from N-terminus to C-terminus asfollows: Agent-CM1-CM2-AB, AB-CM2-CM1-Agent, Agent-CM2-CM1-AB, orAB-CM1-CM2-Agent. In some embodiments, the activatable antibody includesa linking peptide between the agent and CM1. In some embodiments, theactivatable antibody includes a linking peptide between CM1 and CM2. Insome embodiments, the activatable antibody includes a linking peptidebetween CM2 and AB. In some embodiments, the activatable antibodyincludes a linking peptide between the agent and CM1 and a linkingpeptide between CM2 and AB. In some embodiments, the activatableantibody includes a linking peptide between agent and CM1 and a linkingpeptide between CM1 and CM2. In some embodiments, the activatableantibody includes a linking peptide between CM1 and CM2 and a linkingpeptide between CM2 and AB. In some embodiments, the activatableantibody includes a linking peptide between agent and CM1, a linkingpeptide between CM1 and CM2, and a linking peptide between CM2 and AB.

In some embodiments, the activatable antibody includes at least a firstCM that includes a substrate for at least one matrix metalloprotease(MMP) and a second CM that includes a substrate sequence. Exemplarysubstrates for the second CM (CM2) include but are not limited tosubstrates cleavable by one or more of the following enzymes orproteases listed in Table 7.

In some embodiments, the CM2 is selected for use with a specificprotease. In some embodiments, the CM2 is a substrate for at least oneprotease selected from the group consisting of a matrix metalloprotease(MMP), a neutrophil elastase, u-type plasminogen activator (uPA, alsoreferred to as urokinase), legumain, matriptase (also referred to hereinas MT-SP1 or MTSP1), thrombin, a cysteine protease such as a cathepsin,ADAM17, BMP-1, HtrA1, and a TMPRSS such as TMPRSS3 or TMPRSS4.

In some embodiments, the CM2 is a substrate for a neutrophil elastase.In some embodiments, the CM2 is a substrate for uPA. In someembodiments, the CM2 is a substrate for legumain. In some embodiments,the CM2 is a substrate for matriptase. In some embodiments, the CM2 is asubstrate for thrombin. In some embodiments, the CM2 is a substrate fora cysteine protease. In some embodiments, the CM2 is a substrate for acathepsin. In some embodiments, the CM2 is a substrate for ADAM17. Insome embodiments, the CM2 is a substrate for BMP-1. In some embodiments,the CM2 is a substrate for HtrA1. In some embodiments, the CM2 is asubstrate for a TMPRSS. In some embodiments, the CM2 is a substrate forTMPRSS3. In some embodiments, the CM2 is a substrate for TMPRSS4.

For example, suitable CM2 are cleaved by at least one protease andinclude the sequence TGRGPSWV (SEQ ID NO: 34); SARGPSRW (SEQ ID NO: 35);TARGPSFK (SEQ ID NO: 36); LSGRSDNH (SEQ ID NO: 37); GGWHTGRN (SEQ ID NO:38); HTGRSGAL (SEQ ID NO: 39); PLTGRSGG (SEQ ID NO: 40); AARGPAIH (SEQID NO: 41); RGPAFNPM (SEQ ID NO: 42); SSRGPAYL (SEQ ID NO: 43); RGPATPIM(SEQ ID NO: 44); RGPA (SEQ ID NO: 45); GGQPSGMWGW (SEQ ID NO: 46);FPRPLGITGL (SEQ ID NO: 47); VHMPLGFLGP (SEQ ID NO: 48); SPLTGRSG (SEQ IDNO: 49); SAGFSLPA (SEQ ID NO: 126); LAPLGLQRR (SEQ ID NO: 50); SGGPLGVR(SEQ ID NO: 51); PLGL (SEQ ID NO: 52); GPRSFGL (SEQ ID NO: 315) and/orGPRSFG (SEQ ID NO: 316).

In some embodiments, the CM2 comprises the amino acid sequence TGRGPSWV(SEQ ID NO: 34). In some embodiments, the CM2 comprises the amino acidsequence SARGPSRW (SEQ ID NO: 35). In some embodiments, the CM2comprises the amino acid sequence TARGPSFK (SEQ ID NO: 36). In someembodiments, the CM2 comprises the amino acid sequence LSGRSDNH (SEQ IDNO: 37). In some embodiments, the CM2 comprises the amino acid sequenceGGWHTGRN (SEQ ID NO: 38). In some embodiments, the CM2 comprises theamino acid sequence HTGRSGAL (SEQ ID NO: 39). In some embodiments, theCM2 comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 40). In someembodiments, the CM2 comprises the amino acid sequence AARGPAIH (SEQ IDNO: 41). In some embodiments, the CM2 comprises the amino acid sequenceRGPAFNPM (SEQ ID NO: 42). In some embodiments, the CM2 comprises theamino acid sequence SSRGPAYL (SEQ ID NO: 43). In some embodiments, theCM2 comprises the amino acid sequence RGPATPIM (SEQ ID NO: 44). In someembodiments, the CM2 comprises the amino acid sequence RGPA (SEQ ID NO:45). In some embodiments, the CM2 comprises the amino acid sequenceGGQPSGMWGW (SEQ ID NO: 46). In some embodiments, the CM2 comprises theamino acid sequence FPRPLGITGL (SEQ ID NO: 47). In some embodiments, theCM2 comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 48). Insome embodiments, the CM2 comprises the amino acid sequence SPLTGRSG(SEQ ID NO: 49). In some embodiments, the CM2 comprises the amino acidsequence LAPLGLQRR (SEQ ID NO: 50). In some embodiments, the CM2comprises the amino acid sequence SGGPLGVR (SEQ ID NO: 51). In someembodiments, the CM2 comprises the amino acid sequence PLGL (SEQ ID NO:52). In some embodiments, the CM2 comprises the amino acid sequenceGPRSFGL (SEQ ID NO: 315). In some embodiments, the CM2 comprises theamino acid sequence GPRSFG (SEQ ID NO: 316).

In some embodiments, the CM2 is a substrate for at least one MMP. Insome embodiments, the CM2 is a substrate for at least one MMP listed inthe Table 7. In some embodiments, the CM2 is a substrate for MMP9. Insome embodiments, the CM2 is a substrate for MMP14. In some embodiments,CM1 is substrate for a first MMP, and CM2 is a substrate for a secondMMP, where the first MMP and the second MMP are different MMPs. In someembodiments, CM1 is a first substrate sequence for a MMP, and CM2 is asecond substrate for the same MMP, where the CM1 and CM2 have differentsubstrate sequences. In some embodiments, the CM2 is a substrate for twoor more MMPs. In some embodiments, the CM2 is a substrate for at leastMMP9 or MMP14. In some embodiments, the CM2 is a substrate for two ormore MMPs. In some embodiments, the CM2 is a substrate for at least MMP9and MMP14. In some embodiments, CM1 and CM2 are both substrates forMMP9. In some embodiments, CM1 and CM2 are both substrates for MMP14. Insome embodiments, CM1 is a substrate for MMP9 and CM2 is a substrate forMMP14. In some embodiments, CM1 is a substrate for MMP14 and CM2 is asubstrate for MMP9.

In some embodiments, at least one of CM1 and/or CM2 is a substrate foran MMP and includes the sequence ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQID NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17); PVGYTSSL(SEQ ID NO: 18); DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ ID NO: 20);RPSPMWAY (SEQ ID NO: 21); WATPRPMR (SEQ ID NO: 22); FRLLDWQW (SEQ ID NO:23); LKAAPRWA (SEQ ID NO: 24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQID NO: 26); MGLFSEAG (SEQ ID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG(SEQ ID NO: 29); LAAPLGLL (SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31);LLAPSHRA (SEQ ID NO: 32), PAGLWLDP (SEQ ID NO: 33); and/or ISSGLSS (SEQID NO: 159).

In some embodiments, the first cleaving agent and the second cleavingagent are the same protease, and the first CM and the second CM aredifferent substrates for the enzyme. In some embodiments, the firstcleaving agent and the second cleaving agent are different proteases. Insome embodiments, the first cleaving agent and the second cleaving agentare co-localized in the target tissue. In some embodiments, the first CMand the second CM are cleaved by at least one cleaving agent in thetarget tissue.

In some embodiments, the agent conjugated to the AB is a therapeuticagent. In some embodiments, the agent is an antineoplastic agent. Insome embodiments, the agent is a toxin or fragment thereof. As usedherein, a fragment of a toxin is a fragment that retains toxic activity.In some embodiments, the agent is conjugated to the AB via a cleavablelinker. In some embodiments, the agent is conjugated to the AB via alinker that includes at least one MMP-cleavable substrate sequence. Insome embodiments, the agent is conjugated to the AB via a noncleavablelinker. In some embodiments, the agent is a microtubule inhibitor. Insome embodiments, the agent is a nucleic acid damaging agent, such as aDNA alkylator or DNA intercalator, or other DNA damaging agent. In someembodiments, the agent is an agent selected from the group listed inTable 3. In some embodiments, the agent is a dolastatin. In someembodiments, the agent is an auristatin or derivative thereof. In someembodiments, the agent is auristatin E or a derivative thereof. In someembodiments, the agent is monomethyl auristatin E (MMAE). In someembodiments, the agent is monomethyl auristatin D (MMAD). In someembodiments, the agent is a maytansinoid or maytansinoid derivative. Insome embodiments, the agent is DM1 or DM4. In some embodiments, theagent is a duocarmycin or derivative thereof. In some embodiments, theagent is a calicheamicin or derivative thereof. In some embodiments, theagent is a pyrrolobenzodiazepine.

In some embodiments, the agent is an anti-inflammatory agent.

In some embodiments, the antibody also includes a detectable moiety. Insome embodiments, the detectable moiety is a diagnostic agent.

In some embodiments, the conjugated antibody and/or conjugatedactivatable antibody includes a detectable label. In some embodiments,the detectable label includes an imaging agent, a contrasting agent, anenzyme, a fluorescent label, a chromophore, a dye, one or more metalions, or a ligand-based label. In some embodiments, the imaging agentcomprises a radioisotope. In some embodiments, the radioisotope isindium or technetium. In some embodiments, the contrasting agentcomprises iodine, gadolinium or iron oxide. In some embodiments, theenzyme comprises horseradish peroxidase, alkaline phosphatase, orβ-galactosidase. In some embodiments, the fluorescent label comprisesyellow fluorescent protein (YFP), cyan fluorescent protein (CFP), greenfluorescent protein (GFP), modified red fluorescent protein (mRFP), redfluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europiumderivative. In some embodiments, the luminescent label comprises anN-methylacrydium derivative. In some embodiments, the label comprises anAlexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In someembodiments, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

In some embodiments, the AB of the antibody naturally contains one ormore disulfide bonds. In some embodiments, the AB can be engineered toinclude one or more disulfide bonds.

In some embodiments, the antibody and/or conjugated antibody ismonospecific. In some embodiments, the antibody and/or conjugatedantibody is multispecific, referred to herein as multispecificantibodies and/or conjugated multispecific antibodies. In someembodiments, the multispecific antibody and/or conjugated multispecificantibody is bispecific or trifunctional. In some embodiments, theantibody and/or conjugated antibody is formulated as part of apro-Bispecific T Cell Engager (pro-BITE) molecule. In some embodiments,the antibody and/or conjugated antibody is formulated as part of apro-Chimeric Antigen Receptor (pro-CAR) modified T cell or otherengineered receptor.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody is monospecific. In some embodiments, theactivatable antibody and/or conjugated activatable antibody ismultispecific, referred to herein as multispecific activatableantibodies and/or conjugated multispecific activatable antibodies. Asused herein, terms such as “activatable antibody” and all grammaticalvariations thereof, unless otherwise noted, are intended to encompass,but are not limited to embodiments where the activatable antibody is amultispecific activatable antibody of the disclosure. As used herein,terms such as “conjugated activatable antibody” and all grammaticalvariations thereof, unless otherwise noted, are intended to encompass,but are not limited to embodiments where the conjugated activatableantibody is a conjugated multispecific activatable antibody of thedisclosure. In some embodiments, the multispecific activatable antibodyand/or conjugated multispecific activatable antibody is bispecific ortrifunctional. In some embodiments, the activatable antibody and/orconjugated activatable antibody is formulated as part of apro-Bispecific T Cell Engager (pro-BITE) molecule. In some embodiments,the activatable antibody and/or conjugated activatable antibody isformulated as part of a pro-Chimeric Antigen Receptor (pro-CAR) modifiedT cell or other engineered receptor.

In some embodiments, the activatable antibodies, conjugated activatableantibodies, multispecific activatable antibodies, and/or conjugatedmultispecific activatable antibodies described herein are used inconjunction with one or more additional agents or a combination ofadditional agents. Suitable additional agents include currentpharmaceutical and/or surgical therapies for an intended application,such as, for example, cancer. For example, the activatable antibodies,conjugated activatable antibodies, multispecific activatable antibodies,and/or conjugated multispecific activatable antibodies can be used inconjunction with an additional chemotherapeutic or anti-neoplasticagent.

The activatable antibodies described herein in an activated state bind agiven target and include (i) an antibody or an antigen binding fragmentthereof (AB) that specifically binds to the target; (ii) a maskingmoiety (MM) that inhibits the binding of the AB to the target in anuncleaved state; and (c) a cleavable moiety (CM) coupled to the AB,wherein the CM is a polypeptide that functions as a substrate for amatrix metalloprotease.

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM.

In some embodiments, the activatable antibody comprises a linkingpeptide between the MM and the CM.

In some embodiments, the activatable antibody comprises a linkingpeptide between the CM and the AB.

In some embodiments, the activatable antibody comprises a first linkingpeptide (LP1) and a second linking peptide (LP2), and the activatableantibody in the uncleaved state has the structural arrangement fromN-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.

In some embodiments, each of LP1 and LP2 is a peptide of about 1 to 20amino acids in length.

In some embodiments, the two linking peptides need not be identical toeach other.

In some embodiments, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 1) and (GGGS)_(n) (SEQ ID NO: 2), where n is aninteger of at least one.

In some embodiments, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of GGSG (SEQ ID NO: 3),GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG(SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8).

In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG(SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID NO:11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO: 156), orGSSGGSGGSGS (SEQ ID NO: 157).

In some embodiments, LP2 comprises the amino acid sequence GSS, GGS,GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).

In some embodiments, the AB has an equilibrium dissociation constant ofabout 100 nM or less for binding to the target.

In some embodiments, the activatable antibody includes an antibody orantigen-binding fragment thereof that specifically binds a target. Insome embodiments, the antibody or immunologically active fragmentthereof that binds the target is a monoclonal antibody, domain antibody,single chain, Fab fragment, a F(ab)₂ fragment, a scFv, a scab, a dAb, asingle domain heavy chain antibody, or a single domain light chainantibody. In some embodiments, such an antibody or immunologicallyactive fragment thereof that binds the target is a mouse, other rodent,chimeric, humanized or fully human monoclonal antibody.

In some embodiments, the activatable antibody is a multispecificactivatable antibody. The multispecific activatable antibodies providedherein are multispecific antibodies that recognize two or more differentantigens or epitopes and that include at least one masking moiety (MM)linked to at least one antigen- or epitope-binding domain of themultispecific antibody such that coupling of the MM reduces the abilityof the antigen- or epitope-binding domain to bind its target. In someembodiments, the MM is coupled to the antigen- or epitope-binding domainof the multispecific antibody via a cleavable moiety (CM) that functionsas a substrate for at least one MMP protease. The activatablemultispecific antibodies provided herein are stable in circulation,activated at intended sites of therapy and/or diagnosis but not innormal, i.e., healthy tissue, and, when activated, exhibit binding to atarget that is at least comparable to the corresponding, unmodifiedmultispecific antibody.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and that containsa combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2sequence, and the VH CDR3 sequence is selected from a VH CDR1 thatsequence includes at least the amino acid sequence SYAMS (SEQ ID NO:498); a VH CD2 sequence that includes at least the amino acid sequenceSIDPEGRQTYYADSVKG (SEQ ID NO: 499); a VH CDR3 sequence that includes atleast the amino acid sequence DIGGRSAFDY (SEQ ID NO: 500), andcombinations thereof.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and that containsa combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequencethat includes at least the amino acid sequence RASQSISSY (SEQ ID NO:501); a VL CDR2 sequence that includes at least the amino acid sequenceAASSLQS (SEQ ID NO: 502); a VL CDR3 sequence that includes at least theamino acid sequence QQTVVAPPL (SEQ ID NO: 503), and combinationsthereof.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and that containsa combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequencethat includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequence SYAMS(SEQ ID NO: 498); a VH CD2 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 499); a VH CDR3sequence that includes a sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence DIGGRSAFDY (SEQ ID NO: 500), and combinations thereof.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and that containsa combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one of the VL CDR1 sequence, the VL CDR2sequence, and the VL CDR3 sequence is selected from a VL CDR1 sequencethat includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceRASQSISSY (SEQ ID NO: 501); a VL CDR2 sequence that includes a sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence AASSLQS (SEQ ID NO: 502); anda VL CDR3 sequence that includes a sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the aminoacid sequence QQTVVAPPL (SEQ ID NO: 503), and combinations thereof.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and that containsa combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence includes at least the amino acidsequence SYAMS (SEQ ID NO: 498); the VH CD2 sequence includes at leastthe amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 499); the VH CDR3sequence includes at least the amino acid sequence DIGGRSAFDY (SEQ IDNO: 500); the VL CDR1 sequence includes at least the amino acid sequenceRASQSISSY (SEQ ID NO: 501); the VL CDR2 sequence includes at least theamino acid sequence AASSLQS (SEQ ID NO: 502); and the VL CDR3 sequenceincludes at least the amino acid sequence QQTVVAPPL (SEQ ID NO: 503).

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds a Jagged target, e.g., Jagged 1 and/or Jagged 2, and that containsa combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence SYAMS (SEQ ID NO: 498); the VH CD2 sequenceincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequenceSIDPEGRQTYYADSVKG (SEQ ID NO: 499); the VH CDR3 sequence includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:500); the VL CDR1 sequence includes a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence RASQSISSY (SEQ ID NO: 501); the VL CDR2 sequenceincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQID NO: 502); and the VL CDR3 sequence includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence QQTVVAPPL (SEQ ID NO: 503).

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds Epidermal Growth Factor Receptor (EGFR) and that contains acombination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequencethat includes at least the amino acid sequence NYGVH (SEQ ID NO: 504); aVH CD2 sequence that includes at least the amino acid sequenceVIWSGGNTDYNTPFTS (SEQ ID NO: 505); a VH CDR3 sequence that includes atleast the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 506); andcombinations thereof.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds EGFR and that contains a combination of a VL CDR1 sequence, a VLCDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VLCDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence isselected from a VL CDR1 sequence that includes at least the amino acidsequence RASQSIGTNIH (SEQ ID NO: 507); a VL CDR2 sequence that includesat least the amino acid sequence KYASESIS (SEQ ID NO: 508); and a VLCDR3 sequence that includes at least the amino acid sequence QQNNNWPTT(SEQ ID NO: 509), and combinations thereof.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds EGFR and that contains a combination of a VH CDR1 sequence, a VHCDR2 sequence, and a VH CDR3 sequence, wherein at least one of the VHCDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence isselected from a VH CDR1 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence NYGVH (SEQ ID NO: 504); a VH CD2 sequencethat includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceVIWSGGNTDYNTPFTS (SEQ ID NO: 505); a VH CDR3 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO:506); and combinations thereof.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds EGFR and that contains a combination of a VL CDR1 sequence, a VLCDR2 sequence, and a VL CDR3 sequence, wherein at least one of the VLCDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence isselected from a VL CDR1 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence RASQSIGTNIH (SEQ ID NO: 507); a VL CDR2sequence that includes a sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence KYASESIS (SEQ ID NO: 508); and a VL CDR3 sequence that includesa sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO:509), and combinations thereof.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds EGFR and that contains a combination of a VH CDR1 sequence, a VHCDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence includesat least the amino acid sequence NYGVH (SEQ ID NO: 504); the VH CD2sequence includes at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQID NO: 505); the VH CDR3 sequence includes at least the amino acidsequence ALTYYDYEFAY (SEQ ID NO: 506); the VL CDR1 sequence includes atleast the amino acid sequence RASQSIGTNIH (SEQ ID NO: 507); the VL CDR2sequence includes at least the amino acid sequence KYASESIS (SEQ ID NO:508); and the VL CDR3 sequence includes at least the amino acid sequenceQQNNNWPTT (SEQ ID NO: 509).

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a firstantibody or antigen binding fragment thereof (AB1) that specificallybinds EGFR and that contains a combination of a VH CDR1 sequence, a VHCDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence includesa sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence NYGVH (SEQ ID NO: 504);the VH CD2 sequence includes a sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence VIWSGGNTDYNTPFTS (SEQ ID NO: 505); the VH CDR3 sequenceincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY(SEQ ID NO: 506); the VL CDR1 sequence includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence RASQSIGTNIH (SEQ ID NO: 507); the VL CDR2sequence includes a sequence that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequenceKYASESIS (SEQ ID NO: 508); and the VL CDR3 sequence includes a sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence QQNNNWPTT (SEQ ID NO: 509).

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a heavy chainamino acid sequence selected from the group consisting of SEQ ID NOs:54, 56, 57, 58, 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114. In someembodiments, the activatable antibody and/or conjugated activatableantibody provided herein, including but not limited to a multispecificactivatable antibody and/or conjugated multispecific activatableantibody of the disclosure, includes at least a light chain amino acidsequence selected from the group consisting of SEQ ID NOs: 55, 59, 60,62, 64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, and 113. In some embodiments, theactivatable antibody and/or conjugated activatable antibody providedherein, including but not limited to a multispecific activatableantibody and/or conjugated multispecific activatable antibody of thedisclosure, includes at least a heavy chain amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 54, 56, 57, 58, 61, 63, 65, 68,70, 72, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, and 114 and a light chain amino acid sequenceselected from the group consisting of SEQ ID NOs: 55, 59, 60, 62, 64,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, and 113.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody provided herein, including but not limited to amultispecific activatable antibody and/or conjugated multispecificactivatable antibody of the disclosure, includes at least a heavy chainamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 54, 56, 57, 58, 61, 63, 65, 68, 70,72, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, and 114. In some embodiments, the activatable antibodyand/or conjugated activatable antibody provided herein, including butnot limited to a multispecific activatable antibody and/or conjugatedmultispecific activatable antibody of the disclosure, includes at leasta light chain amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 55, 59, 60, 62, 64,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, and 113. In some embodiments, the activatableantibody and/or conjugated activatable antibody provided herein,including but not limited to a multispecific activatable antibody and/orconjugated multispecific activatable antibody of the disclosure,includes at least a heavy chain amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to anamino acid sequence selected from the group consisting of SEQ ID NOs:54, 56, 57, 58, 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114 and a lightchain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 55, 59, 60, 62, 64, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, and 113.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is no more than the equilibrium dissociationconstant of the AB to the target.

In some embodiments, the MM does not interfere or compete with the ABfor binding to the target in a cleaved state.

In some embodiments, the MM is a polypeptide of about 2 to 40 aminoacids in length. For example, the MM is a polypeptide of up to about 40amino acids in length.

In some embodiments, the MM polypeptide sequence is different from thatof any natural binding partner of the AB. In some embodiments, the MMpolypeptide sequence is no more than 50% identical to any naturalbinding partner of the AB. In some embodiments, the MM polypeptidesequence is no more than 40%, 30%, 25%, 20%, 15%, or 10% identical toany natural binding partner of the AB.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind its target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at leasttwo times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind its target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at leastthree times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind its target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at leastfive times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind its target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least10 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind its target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least20 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind the target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least40 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind the target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least100 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind the target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least1000 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind the target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least10,000 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the MMP protease is co-localized with the target ina tissue, and the MMP cleaves the CM in the activatable antibody whenthe activatable antibody is exposed to the MMP.

In some embodiments, in the presence of the target, the MM reduces theability of the AB to bind the target by at least 90% when the CM isuncleaved, as compared to when the CM is cleaved when assayed in vitrousing a target displacement assay such as, for example, the assaydescribed in PCT Publication Nos. WO 2009/025846 and WO 2010/081173.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least twofold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state (i.e., when the activatable antibody is in the cleavedstate), the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least five-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state (i.e., when the activatable antibody is in the cleavedstate), the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least ten-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state (i.e., when the activatable antibody is in the cleavedstate), the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 20-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state (i.e., when the activatable antibody is in the cleavedstate), the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 40-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state, the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 50-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state, the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 100-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state, the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 200-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state, the AB binds the target.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength.

In some embodiments, the CM is a substrate for at least one matrixmetalloprotease (MMP). Examples of MMPs include MMP1; MMP2; MMP3; MMP7;MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17;MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. In some embodiments, theCM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, andMMP19. In some embodiments, the CM is a substrate for MMP9. In someembodiments, the CM is a substrate for MMP14. In some embodiments, theCM is a substrate for two or more MMPs. In some embodiments, the CM is asubstrate for at least MMP9 and MMP14. In some embodiments, the CMcomprises two or more substrates for the same MMP. In some embodiments,the CM comprises at least two or more MMP9 substrates. In someembodiments, the CM comprises at least two or more MMP14 substrates.

In some embodiments, the CM is a substrate for an MMP and includes thesequence ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID NO: 15); AQNLLGMV(SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17); PVGYTSSL (SEQ ID NO: 18);DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ ID NO: 20); RPSPMWAY (SEQ ID NO:21); WATPRPMR (SEQ ID NO: 22); FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQID NO: 24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG(SEQ ID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29);LAAPLGLL (SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO:32), PAGLWLDP (SEQ ID NO: 33); and/or ISSGLSS (SEQ ID NO: 159).

In some embodiments, the CM comprises the amino acid sequence ISSGLLSS(SEQ ID NO: 14). In some embodiments, the CM comprises the amino acidsequence QNQALRMA (SEQ ID NO: 15). In some embodiments, the CM comprisesthe amino acid sequence AQNLLGMV (SEQ ID NO: 16). In some embodiments,the CM comprises the amino acid sequence STFPFGMF (SEQ ID NO: 17). Insome embodiments, the CM comprises the amino acid sequence PVGYTSSL (SEQID NO: 18). In some embodiments, the CM comprises the amino acidsequence DWLYWPGI (SEQ ID NO: 19). In some embodiments, the CM comprisesthe amino acid sequence MIAPVAYR (SEQ ID NO: 20). In some embodiments,the CM comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 21). Insome embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQID NO: 22). In some embodiments, the CM comprises the amino acidsequence FRLLDWQW (SEQ ID NO: 23). In some embodiments, the CM comprisesthe amino acid sequence LKAAPRWA (SEQ ID NO: 24). In some embodiments,the CM comprises the amino acid sequence GPSHLVLT (SEQ ID NO: 25). Insome embodiments, the CM comprises the amino acid sequence LPGGLSPW (SEQID NO: 26). In some embodiments, the CM comprises the amino acidsequence MGLFSEAG (SEQ ID NO: 27). In some embodiments, the CM comprisesthe amino acid sequence SPLPLRVP (SEQ ID NO: 28). In some embodiments,the CM comprises the amino acid sequence RMHLRSLG (SEQ ID NO: 29). Insome embodiments, the CM comprises the amino acid sequence LAAPLGLL (SEQID NO: 30). In some embodiments, the CM comprises the amino acidsequence AVGLLAPP (SEQ ID NO: 31). In some embodiments, the CM comprisesthe amino acid sequence LLAPSHRA (SEQ ID NO: 32). In some embodiments,the CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 33). Insome embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQID NO: 159).

In some embodiments, the CM is a substrate for at least two proteases.In some embodiments, at least one protease is an MMP and at least oneprotease is selected from the group consisting of those shown in Table7.

In some embodiments, the activatable antibody includes at least a firstCM and a second CM. In some embodiments, the first CM and the second CMare each polypeptides of no more than 15 amino acids long. In someembodiments, the first CM and the second CM in the activatable antibodyhave the structural arrangement from N-terminus to C-terminus as followsin the uncleaved state: MM-CM 1-CM2-AB, AB-CM2-CM 1-MM, MM-CM2-CM1-AB,or AB-CM1-CM2-MM. In some embodiments, the activatable antibody includesa linking peptide between MM and CM1. In some embodiments, theactivatable antibody includes a linking peptide between CM1 and CM2. Insome embodiments, the activatable antibody includes a linking peptidebetween CM2 and AB. In some embodiments, the activatable antibodyincludes a linking peptide between MM and CM1 and a linking peptidebetween CM2 and AB. In some embodiments, the activatable antibodyincludes a linking peptide between MM and CM1 and a linking peptidebetween CM1 and CM2. In some embodiments, the activatable antibodyincludes a linking peptide between CM1 and CM2 and a linking peptidebetween CM2 and AB. In some embodiments, the activatable antibodyincludes a linking peptide between MM and CM1, a linking peptide betweenCM1 and CM2, and a linking peptide between CM2 and AB.

In some embodiments, the activatable antibody includes at least a firstCM that includes a substrate for at least one matrix metalloprotease(MMP) and a second CM that includes a substrate sequence. Exemplarysubstrates for the second CM (CM2) include but are not limited tosubstrates cleavable by one or more of the following enzymes orproteases listed in Table 7.

In some embodiments, the CM2 is selected for use with a specificprotease. In some embodiments, the CM2 is a substrate for at least oneprotease selected from the group consisting of a matrix metalloprotease(MMP), a neutrophil elastase, u-type plasminogen activator (uPA, alsoreferred to as urokinase), legumain, matriptase (MT-SP1), thrombin, acysteine protease such as a cathepsin, ADAM17, BMP-1, HtrA1, and aTMPRSS such as TMPRSS3 or TMPRSS4.

In some embodiments, the CM2 is a substrate for a neutrophil elastase.In some embodiments, the CM2 is a substrate for uPA. In someembodiments, the CM2 is a substrate for legumain. In some embodiments,the CM2 is a substrate for matriptase. In some embodiments, the CM2 is asubstrate for thrombin. In some embodiments, the CM2 is a substrate fora cysteine protease. In some embodiments, the CM2 is a substrate for acathepsin. In some embodiments, the CM2 is a substrate for ADAM17. Insome embodiments, the CM2 is a substrate for BMP-1. In some embodiments,the CM2 is a substrate for HtrA1. In some embodiments, the CM2 is asubstrate for a TMPRSS. In some embodiments, the CM2 is a substrate forTMPRSS3. In some embodiments, the CM2 is a substrate for TMPRSS4.

For example, suitable CM2 are cleaved by at least one protease andinclude the sequence TGRGPSWV (SEQ ID NO: 34); SARGPSRW (SEQ ID NO: 35);TARGPSFK (SEQ ID NO: 36); LSGRSDNH (SEQ ID NO: 37); GGWHTGRN (SEQ ID NO:38); HTGRSGAL (SEQ ID NO: 39); PLTGRSGG (SEQ ID NO: 40); AARGPAIH (SEQID NO: 41); RGPAFNPM (SEQ ID NO: 42); SSRGPAYL (SEQ ID NO: 43); RGPATPIM(SEQ ID NO: 44); RGPA (SEQ ID NO: 45); GGQPSGMWGW (SEQ ID NO: 46);FPRPLGITGL (SEQ ID NO: 47); VHMPLGFLGP (SEQ ID NO: 48); SPLTGRSG (SEQ IDNO: 49); SAGFSLPA (SEQ ID NO: 126); LAPLGLQRR (SEQ ID NO: 50); SGGPLGVR(SEQ ID NO: 51); PLGL (SEQ ID NO: 52); GPRSFGL (SEQ ID NO: 315) and/orGPRSFG (SEQ ID NO: 316).

In some embodiments, the CM2 comprises the amino acid sequence TGRGPSWV(SEQ ID NO: 34). In some embodiments, the CM2 comprises the amino acidsequence SARGPSRW (SEQ ID NO: 35). In some embodiments, the CM2comprises the amino acid sequence TARGPSFK (SEQ ID NO: 36). In someembodiments, the CM2 comprises the amino acid sequence LSGRSDNH (SEQ IDNO: 37). In some embodiments, the CM2 comprises the amino acid sequenceGGWHTGRN (SEQ ID NO: 38). In some embodiments, the CM2 comprises theamino acid sequence HTGRSGAL (SEQ ID NO: 39). In some embodiments, theCM2 comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 40). In someembodiments, the CM2 comprises the amino acid sequence AARGPAIH (SEQ IDNO: 41). In some embodiments, the CM2 comprises the amino acid sequenceRGPAFNPM (SEQ ID NO: 42). In some embodiments, the CM2 comprises theamino acid sequence SSRGPAYL (SEQ ID NO: 43). In some embodiments, theCM2 comprises the amino acid sequence RGPATPIM (SEQ ID NO: 44). In someembodiments, the CM2 comprises the amino acid sequence RGPA (SEQ ID NO:45). In some embodiments, the CM2 comprises the amino acid sequenceGGQPSGMWGW (SEQ ID NO: 46). In some embodiments, the CM2 comprises theamino acid sequence FPRPLGITGL (SEQ ID NO: 47). In some embodiments, theCM2 comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 48). Insome embodiments, the CM2 comprises the amino acid sequence SPLTGRSG(SEQ ID NO: 49). In some embodiments, the CM2 comprises the amino acidsequence LAPLGLQRR (SEQ ID NO: 50). In some embodiments, the CM2comprises the amino acid sequence SGGPLGVR (SEQ ID NO: 51). In someembodiments, the CM2 comprises the amino acid sequence PLGL (SEQ ID NO:52). In some embodiments, the CM2 comprises the amino acid sequenceGPRSFGL (SEQ ID NO: 315). In some embodiments, the CM2 comprises theamino acid sequence GPRSFG (SEQ ID NO: 316)

In some embodiments, the CM2 is a substrate for at least one MMP. Insome embodiments, the CM2 is a substrate for at least one MMP listed inthe Table 7. In some embodiments, the CM2 is a substrate for MMP9. Insome embodiments, the CM2 is a substrate for MMP14. In some embodiments,CM1 is substrate for a first MMP, and CM2 is a substrate for a secondMMP, where the first MMP and the second MMP are different MMPs. In someembodiments, CM1 is a first substrate sequence for a MMP, and CM2 is asecond substrate for the same MMP, where the CM1 and CM2 have differentsubstrate sequences. In some embodiments, the CM2 is a substrate for twoor more MMPs. In some embodiments, the CM2 is a substrate for at leastMMP9 or MMP14. In some embodiments, the CM2 is a substrate for two ormore MMPs. In some embodiments, the CM2 is a substrate for at least MMP9and MMP14. In some embodiments, CM1 and CM2 are both substrates forMMP9. In some embodiments, CM1 and CM2 are both substrates for MMP14. Insome embodiments, CM1 is a substrate for MMP9 and CM2 is a substrate forMMP14. In some embodiments, CM1 is a substrate for MMP14 and CM2 is asubstrate for MMP9.

In some embodiments, at least one of CM1 and/or CM2 is a substrate foran MMP and includes the sequence ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQID NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17); PVGYTSSL(SEQ ID NO: 18); DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ ID NO: 20);RPSPMWAY (SEQ ID NO: 21); WATPRPMR (SEQ ID NO: 22); FRLLDWQW (SEQ ID NO:23); LKAAPRWA (SEQ ID NO: 24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQID NO: 26); MGLFSEAG (SEQ ID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG(SEQ ID NO: 29); LAAPLGLL (SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31);LLAPSHRA (SEQ ID NO: 32), PAGLWLDP (SEQ ID NO: 33); and/or ISSGLSS (SEQID NO: 159).

In some embodiments, the first cleaving agent and the second cleavingagent are the same matrix metalloprotease, and the first CM and thesecond CM are different substrates for the enzyme. In some embodiments,the first cleaving agent and the second cleaving agent are differentproteases, where at least one protease is an MMP. In some embodiments,the first cleaving agent and the second cleaving agent are co-localizedin the target tissue. In some embodiments, the first CM and the secondCM are cleaved by at least one cleaving agent in the target tissue.

In some embodiments, the activatable antibody is exposed to and cleavedby a MMP such that, in the activated or cleaved state, the activatedantibody includes a light chain amino acid sequence that includes atleast a portion of LP2 and/or CM sequence after the MMP has cleaved theCM.

In some embodiments, the CM comprises the non-prime side of the proteasecleavage site; that is, the CM comprises at least the P1 and P2 aminoacids, and in some embodiments, comprises the P1, P2 and P3 amino acidsand in some embodiments, comprises the P1, P2, P3, and P4 amino acids.In some embodiments, the CM comprises the non-prime side and the primeside of the protease cleavage site. In some embodiments, the CMcomprises the non-prime side but lacks at least part of the prime sideof the protease cleavage site. In some embodiments, the CM comprises thenon-prime side but lacks the prime side of the protease cleavage site.Such a CM can be linked directly or through a linker to an antibody orother molecule as disclosed herein, such as, but not limited to, adetection moiety.

In some embodiments, the activatable antibody is an anti-EGFRactivatable antibody that includes at least an AB that is or is derivedfrom cetuximab or panitumumab; a MM comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 160, 167-200, and 497;and a CM comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 14-33, and 159. In some embodiments, theactivatable antibody is an anti-EGFR activatable antibody that includesat least an AB that is or is derived from cetuximab or panitumumab; a MMcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 160, 167-200, and 497; and a CM comprising an amino acidsequence selected from the group consisting of the sequences presentedin Tables 8A-8M. In some embodiments, the anti-EGFR activatable antibodyalso includes a first linking peptide (LP1) and a second linking peptide(LP2), and the activatable antibody in the uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, each of LP1and LP2 is a peptide of about 1 to 20 amino acids in length. In someembodiments, the two linking peptides need not be identical to eachother. In some embodiments, at least one of LP1 or LP2 comprises anamino acid sequence selected from the group consisting of (GS)_(n),(GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 1) and (GGGS)_(n) (SEQ ID NO: 2),where n is an integer of at least one. In some embodiments, at least oneof LP1 or LP2 comprises an amino acid sequence selected from the groupconsisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ IDNO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ IDNO: 8). In some embodiments, LP1 comprises the amino acid sequenceGSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS(SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ IDNO: 156), or GSSGGSGGSGS (SEQ ID NO: 157). In some embodiments, LP2comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT(SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).

In some embodiments, the activatable antibody is an anti-EGFRactivatable antibody that includes at least an AB comprising a heavychain amino acid sequence comprising the VH CDR1 sequence of SEQ ID NO:504, the VH CDR2 sequence of SEQ ID NO: 505, the VH CDR3 sequence of SEQID NO: 506, the VL CDR1 sequence of SEQ ID NO: 507, the VL CDR2 sequenceof SEQ ID NO: 508, and the VL CDR2 sequence of SEQ ID NO: 509; a MMcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 160, 167-200, and 497; and a CM comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 14-33, and159. In some embodiments, the activatable antibody is an anti-EGFRactivatable antibody that includes at least an AB comprising a heavychain amino acid sequence comprising the VH CDR1 sequence of SEQ ID NO:504, the VH CDR2 sequence of SEQ ID NO: 505, the VH CDR3 sequence of SEQID NO: 506, the VL CDR1 sequence of SEQ ID NO: 507, the VL CDR2 sequenceof SEQ ID NO: 508, and the VL CDR2 sequence of SEQ ID NO: 509; a MMcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 160, 167-200, and 497; and a CM comprising an amino acidsequence selected from the group consisting of the sequences presentedin Tables 8A-8M. In some embodiments, the anti-EGFR activatable antibodyalso includes a first linking peptide (LP1) and a second linking peptide(LP2), and the activatable antibody in the uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, each of LP1and LP2 is a peptide of about 1 to 20 amino acids in length. In someembodiments, the two linking peptides need not be identical to eachother. In some embodiments, at least one of LP1 or LP2 comprises anamino acid sequence selected from the group consisting of (GS)_(n),(GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 1) and (GGGS)_(n) (SEQ ID NO: 2),where n is an integer of at least one. In some embodiments, at least oneof LP1 or LP2 comprises an amino acid sequence selected from the groupconsisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ IDNO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ IDNO: 8). In some embodiments, LP1 comprises the amino acid sequenceGSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS(SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ IDNO: 156), or GSSGGSGGSGS (SEQ ID NO: 157). In some embodiments, LP2comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT(SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).

In some embodiments, the activatable antibody is an anti-EGFRactivatable antibody that includes at least an AB comprising the heavychain amino acid sequence of SEQ ID NO: 56, 57 or 58 and the light chainamino acid sequence of SEQ ID NO: 59; a MM comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 160, 167-200,and 497; and a CM comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 14-33, and 159. In some embodiments, theactivatable antibody is an anti-EGFR activatable antibody that includesat least an AB comprising the heavy chain amino acid sequence of SEQ IDNO: 56, 57 or 58 and the light chain amino acid sequence of SEQ ID NO:59; a MM comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 160, 167-200, and 497; and a CM comprising anamino acid sequence selected from the group consisting of the sequencespresented in Tables 8A-8M. In some embodiments, the anti-EGFRactivatable antibody also includes a first linking peptide (LP1) and asecond linking peptide (LP2), and the activatable antibody in theuncleaved state has the structural arrangement from N-terminus toC-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP 1-MM. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length. In some embodiments, the two linking peptides need notbe identical to each other. In some embodiments, at least one of LP1 orLP2 comprises an amino acid sequence selected from the group consistingof (GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 1) and (GGGS)_(n) (SEQID NO: 2), where n is an integer of at least one. In some embodiments,at least one of LP1 or LP2 comprises an amino acid sequence selectedfrom the group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4),GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), andGSSSG (SEQ ID NO: 8). In some embodiments, LP1 comprises the amino acidsequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10),GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155),GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO: 157). In someembodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).

In some embodiments, the activatable antibody is an anti-EGFRactivatable antibody that includes at least an AB comprising the heavychain amino acid sequence of SEQ ID NO: 56 and the light chain aminoacid sequence of SEQ ID NO: 59; a MM comprising the amino acid sequenceof SEQ ID NO: 160; and a CM comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 14-33, and 159. In someembodiments, the activatable antibody is an anti-EGFR activatableantibody that includes at least an AB comprising the heavy chain aminoacid sequence of SEQ ID NO: 56 and the light chain amino acid sequenceof SEQ ID NO: 59; a MM comprising the amino acid sequence of SEQ ID NO:160; and a CM comprising an amino acid sequence selected from the groupconsisting of the sequences presented in Tables 8A-8M. In someembodiments, the anti-EGFR activatable antibody also includes a firstlinking peptide (LP1) and a second linking peptide (LP2), and theactivatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-ABor AB-LP2-CM-LP1-MM. In some embodiments, each of LP1 and LP2 is apeptide of about 1 to 20 amino acids in length. In some embodiments, thetwo linking peptides need not be identical to each other. In someembodiments, at least one of LP1 or LP2 comprises an amino acid sequenceselected from the group consisting of (GS)_(n), (GGS)_(n), (GSGGS)_(n)(SEQ ID NO: 1) and (GGGS)_(n) (SEQ ID NO: 2), where n is an integer ofat least one. In some embodiments, at least one of LP1 or LP2 comprisesan amino acid sequence selected from the group consisting of GGSG (SEQID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8). In some embodiments,LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9),GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11),GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO: 156), orGSSGGSGGSGS (SEQ ID NO: 157). In some embodiments, LP2 comprises theamino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO:12) or GSSG (SEQ ID NO: 13).

In some embodiments, the activatable antibody is an anti-EGFRactivatable antibody that includes at least an AB comprising a heavychain amino acid sequence comprising the VH CDR1 sequence of SEQ ID NO:504, the VH CDR2 sequence of SEQ ID NO: 505, the VH CDR3 sequence of SEQID NO: 506, the VL CDR1 sequence of SEQ ID NO: 507, the VL CDR2 sequenceof SEQ ID NO: 508, and the VL CDR2 sequence of SEQ ID NO: 509; a MMcomprising the amino acid sequence of SEQ ID NO: 160; and a CMcomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 14-33, and 159. In some embodiments, the activatable antibodyis an anti-EGFR activatable antibody that includes at least an ABcomprising a heavy chain amino acid sequence comprising the VH CDR1sequence of SEQ ID NO: 504, the VH CDR2 sequence of SEQ ID NO: 505, theVH CDR3 sequence of SEQ ID NO: 506, the VL CDR1 sequence of SEQ ID NO:507, the VL CDR2 sequence of SEQ ID NO: 508, and the VL CDR2 sequence ofSEQ ID NO: 509; a MM comprising the amino acid sequence of SEQ ID NO:160; and a CM comprising an amino acid sequence selected from the groupconsisting of the sequences presented in Tables 8A-8M. In someembodiments, the anti-EGFR activatable antibody also includes a firstlinking peptide (LP1) and a second linking peptide (LP2), and theactivatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-ABor AB-LP2-CM-LP 1-MM. In some embodiments, each of LP1 and LP2 is apeptide of about 1 to 20 amino acids in length. In some embodiments, thetwo linking peptides need not be identical to each other. In someembodiments, at least one of LP1 or LP2 comprises an amino acid sequenceselected from the group consisting of (GS)_(n), (GGS)_(n), (GSGGS)_(n)(SEQ ID NO: 1) and (GGGS)_(n) (SEQ ID NO: 2), where n is an integer ofat least one. In some embodiments, at least one of LP1 or LP2 comprisesan amino acid sequence selected from the group consisting of GGSG (SEQID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8). In some embodiments,LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9),GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11),GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO: 156), orGSSGGSGGSGS (SEQ ID NO: 157). In some embodiments, LP2 comprises theamino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO:12) or GSSG (SEQ ID NO: 13).

In some embodiments, the activatable antibody is an anti-Jaggedactivatable antibody that includes at least an AB comprising the heavychain amino acid sequence of SEQ ID NO: 61, 63, 65, 68, 70, 72, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, or 114 and the light chain amino acid sequence of SEQ ID NO: 60,62, 64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, or 113; a MM comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 201-263, and496; and a CM comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 14-33, and 159. In some embodiments, theactivatable antibody is an anti-Jagged activatable antibody thatincludes at least an AB comprising the heavy chain amino acid sequenceof SEQ ID NO: 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, or 114 and the lightchain amino acid sequence of SEQ ID NO: 60, 62, 64, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, or 113; a MM comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 201-263, and 496; and a CM comprising anamino acid sequence selected from the group consisting of the sequencespresented in Tables 8A-8M. In some embodiments, the anti-Jaggedactivatable antibody also includes a first linking peptide (LP1) and asecond linking peptide (LP2), and the activatable antibody in theuncleaved state has the structural arrangement from N-terminus toC-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length. In some embodiments, the two linking peptides need notbe identical to each other. In some embodiments, at least one of LP1 orLP2 comprises an amino acid sequence selected from the group consistingof (GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 1) and (GGGS)_(n) (SEQID NO: 2), where n is an integer of at least one. In some embodiments,at least one of LP1 or LP2 comprises an amino acid sequence selectedfrom the group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4),GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), andGSSSG (SEQ ID NO: 8). In some embodiments, LP1 comprises the amino acidsequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10),GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155),GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO: 157). In someembodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).

In some embodiments, the activatable antibody is an anti-Jaggedactivatable antibody that includes at least an AB comprising the heavychain amino acid sequence of SEQ ID NO: 112 and the light chain aminoacid sequence of SEQ ID NO: 111; a MM comprising the amino acid sequenceselected of SEQ ID NO: 217; and a CM comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 14-33, and 159. In someembodiments, the activatable antibody is an anti-Jagged activatableantibody that includes at least an AB comprising the heavy chain aminoacid sequence of SEQ ID NO: 112 and the light chain amino acid sequenceof SEQ ID NO: 111; a MM comprising the amino acid sequence selected ofSEQ ID NO: 217; and a CM comprising an amino acid sequence selected fromthe group consisting of the sequences presented in Tables 8A-8M. In someembodiments, the anti-Jagged activatable antibody also includes a firstlinking peptide (LP1) and a second linking peptide (LP2), and theactivatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-ABor AB-LP2-CM-LP 1-MM. In some embodiments, each of LP1 and LP2 is apeptide of about 1 to 20 amino acids in length. In some embodiments, thetwo linking peptides need not be identical to each other. In someembodiments, at least one of LP1 or LP2 comprises an amino acid sequenceselected from the group consisting of (GS)_(n), (GGS)_(n), (GSGGS)_(n)(SEQ ID NO: 1) and (GGGS)_(n) (SEQ ID NO: 2), where n is an integer ofat least one. In some embodiments, at least one of LP1 or LP2 comprisesan amino acid sequence selected from the group consisting of GGSG (SEQID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8). In some embodiments,LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9),GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11),GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO: 156), orGSSGGSGGSGS (SEQ ID NO: 157). In some embodiments, LP2 comprises theamino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO:12) or GSSG (SEQ ID NO: 13).

In some embodiments, the activatable antibody is an anti-Jaggedactivatable antibody that includes at least an AB comprising a heavychain amino acid sequence comprising the VH CDR1 sequence of SEQ ID NO:498, the VH CDR2 sequence of SEQ ID NO: 499, the VH CDR3 sequence of SEQID NO: 500, the VL CDR1 sequence of SEQ ID NO: 501, the VL CDR2 sequenceof SEQ ID NO: 502, and the VL CDR2 sequence of SEQ ID NO: 503; a MMcomprising the amino acid sequence selected from the group consisting ofSEQ ID NO: 217; and a CM comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 14-33, and 159. In some embodiments,the activatable antibody is an anti-Jagged activatable antibody thatincludes at least an AB comprising a heavy chain amino acid sequencecomprising the VH CDR1 sequence of SEQ ID NO: 498, the VH CDR2 sequenceof SEQ ID NO: 499, the VH CDR3 sequence of SEQ ID NO: 500, the VL CDR1sequence of SEQ ID NO: 501, the VL CDR2 sequence of SEQ ID NO: 502, andthe VL CDR2 sequence of SEQ ID NO: 503; a MM comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 217; and a CMcomprising an amino acid sequence selected from the group consisting ofthe sequences presented in Tables 8A-8M. In some embodiments, theanti-Jagged activatable antibody also includes a first linking peptide(LP1) and a second linking peptide (LP2), and the activatable antibodyin the uncleaved state has the structural arrangement from N-terminus toC-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length. In some embodiments, the two linking peptides need notbe identical to each other. In some embodiments, at least one of LP1 orLP2 comprises an amino acid sequence selected from the group consistingof (GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 1) and (GGGS)_(n) (SEQID NO: 2), where n is an integer of at least one. In some embodiments,at least one of LP1 or LP2 comprises an amino acid sequence selectedfrom the group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4),GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), andGSSSG (SEQ ID NO: 8). In some embodiments, LP1 comprises the amino acidsequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10),GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155),GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO: 157). In someembodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).

In some embodiments, the activatable antibody also includes an agentconjugated to the AB. In some embodiments, the agent is a therapeuticagent. In some embodiments, the agent is an antineoplastic agent. Insome embodiments, the agent is a toxin or a fragment thereof. In someembodiments, the agent is conjugated to the AB via a linker. In someembodiments, the linker is a cleavable linker. In some embodiments, theagent is a microtubule inhibitor. In some embodiments, the agent is anucleic acid damaging agent, such as a DNA alkylator or DNAintercalator, or other DNA damaging agent. In some embodiments, thelinker is a cleavable linker. In some embodiments, the agent isconjugated to the AB via a linker that includes at least oneMMP-cleavable substrate sequence. In some embodiments, the agent is anagent selected from the group listed in Table 3. In some embodiments,the agent is a dolastatin. In some embodiments, the agent is anauristatin or derivative thereof. In some embodiments, the agent isauristatin E or a derivative thereof. In some embodiments, the agent ismonomethyl auristatin E (MMAE). In some embodiments, the agent ismonomethyl auristatin D (MMAD). In some embodiments, the agent is amaytansinoid or maytansinoid derivative. In some embodiments, the agentis DM1 or DM4. In some embodiments, the agent is a duocarmycin orderivative thereof. In some embodiments, the agent is a calicheamicin orderivative thereof. In some embodiments, the agent is apyrrolobenzodiazepine.

In some embodiments, the agent is an anti-inflammatory agent.

In some embodiments, the activatable antibody also includes a detectablemoiety. In some embodiments, the detectable moiety is a diagnosticagent.

In some embodiments, the conjugated antibody includes a detectablelabel. In some embodiments, the detectable label includes an imagingagent, a contrasting agent, an enzyme, a fluorescent label, achromophore, a dye, one or more metal ions, or a ligand-based label. Insome embodiments, the imaging agent comprises a radioisotope. In someembodiments, the radioisotope is indium or technetium. In someembodiments, the contrasting agent comprises iodine, gadolinium or ironoxide. In some embodiments, the enzyme comprises horseradish peroxidase,alkaline phosphatase, or β-galactosidase. In some embodiments, thefluorescent label comprises yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), green fluorescent protein (GFP), modified redfluorescent protein (mRFP), red fluorescent protein tdimer2 (RFPtdimer2), HCRED, or a europium derivative. In some embodiments, theluminescent label comprises an N-methylacrydium derivative. In someembodiments, the label comprises an Alexa Fluor® label, such as AlexFluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-basedlabel comprises biotin, avidin, streptavidin or one or more haptens.

In some embodiments, the activatable antibody also includes a signalpeptide. In some embodiments, the signal peptide is conjugated to theactivatable antibody via a spacer. In some embodiments, the spacer isconjugated to the activatable antibody in the absence of a signalpeptide. In some embodiments, the spacer is joined directly to the MM ofthe activatable antibody. In some embodiments, the spacer is joineddirectly to the MM of the activatable antibody in the structuralarrangement from N-terminus to C-terminus of spacer-MM-CM-AB. An exampleof a spacer joined directly to the N-terminus of MM of the activatableantibody is QGQSGQ (SEQ ID NO: 53). In some embodiments, the spacerincludes at least the amino acid sequence QGQSGQ (SEQ ID NO: 53).

In some embodiments, the AB of the activatable antibody naturallycontains one or more disulfide bonds. In some embodiments, the AB can beengineered to include one or more disulfide bonds.

In some embodiments, the serum half-life of the activatable antibody islonger than that of the corresponding antibody; e.g., the pK of theactivatable antibody is longer than that of the corresponding antibody.In some embodiments, the serum half-life of the activatable antibody issimilar to that of the corresponding antibody. In some embodiments, theserum half-life of the activatable antibody is at least 15 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 12 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 11 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least10 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 9 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 8 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 7 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least6 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 5 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 4 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 3 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least2 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 24 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 20 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 18 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least16 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 14 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 12 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 10 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least8 hours when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 6 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 4 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 3 hours when administered to an organism.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody is monospecific. In some embodiments, theactivatable antibody and/or conjugated activatable antibody ismultispecific, e.g., by way of non-limiting example, bispecific ortrifunctional. In some embodiments, the activatable antibody and/orconjugated activatable antibody is formulated as part of apro-Bispecific T Cell Engager (pro-BITE) molecule. In some embodiments,the activatable antibody and/or conjugated activatable antibody isformulated as part of a pro-Chimeric Antigen Receptor (pro-CAR) modifiedT cell or other engineered receptor.

The disclosure also provides compositions and methods that include anactivatable antibody that includes an antibody or antibody fragment (AB)that specifically binds a given target, where the AB is coupled to amasking moiety (MM) that decreases the ability of the AB to bind itstarget. In some embodiments, the activatable antibody further includes acleavable moiety (CM) that is a substrate for at least one MMP. Thecompositions and methods provided herein enable the attachment of one ormore agents to one or more cysteine residues in the AB withoutcompromising the activity (e.g., the masking, activating or bindingactivity) of the activatable antibody. In some embodiments, thecompositions and methods provided herein enable the attachment of one ormore agents to one or more cysteine residues in the AB without reducingor otherwise disturbing one or more disulfide bonds within the MM. Thecompositions and methods provided herein produce an activatable antibodythat is conjugated to one or more agents, e.g., any of a variety oftherapeutic, diagnostic and/or prophylactic agents, for example, in someembodiments, without any of the agent(s) being conjugated to the MM ofthe activatable antibody. The compositions and methods provided hereinproduce conjugated activatable antibodies in which the MM retains theability to effectively and efficiently mask the AB of the activatableantibody in an uncleaved state. The compositions and methods providedherein produce conjugated activatable antibodies in which theactivatable antibody is still activated, i.e., cleaved, in the presenceof a MMP that can cleave the CM.

The activatable antibodies have at least one point of conjugation for anagent, but in the methods and compositions provided herein less than allpossible points of conjugation are available for conjugation to anagent. In some embodiments, the one or more points of conjugation aresulfur atoms involved in disulfide bonds. In some embodiments, the oneor more points of conjugation are sulfur atoms involved in interchaindisulfide bonds. In some embodiments, the one or more points ofconjugation are sulfur atoms involved in interchain sulfide bonds, butnot sulfur atoms involved in intrachain disulfide bonds. In someembodiments, the one or more points of conjugation are sulfur atoms ofcysteine or other amino acid residues containing a sulfur atom. Suchresidues may occur naturally in the antibody structure or may beincorporated into the antibody by site-directed mutagenesis, chemicalconversion, or mis-incorporation of non-natural amino acids.

Also provided are methods of preparing a conjugate of an activatableantibody having one or more interchain disulfide bonds in the AB and oneor more intrachain disulfide bonds in the MM, and a drug reactive withfree thiols is provided. The method generally includes partiallyreducing interchain disulfide bonds in the activatable antibody with areducing agent, such as, for example, TCEP; and conjugating the drugreactive with free thiols to the partially reduced activatable antibody.As used herein, the term partial reduction refers to situations where anactivatable antibody is contacted with a reducing agent and less thanall disulfide bonds, e.g., less than all possible sites of conjugationare reduced. In some embodiments, less than 99%, 98%, 97%, 96%, 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10% or less than 5% of all possible sites of conjugation arereduced.

In some embodiments, a method of reducing and conjugating an agent,e.g., a drug, to an activatable antibody resulting in selectivity in theplacement of the agent is provided. The method generally includespartially reducing the activatable antibody with a reducing agent suchthat any conjugation sites in the masking moiety or other non-AB portionof the activatable antibody are not reduced, and conjugating the agentto interchain thiols in the AB. The conjugation site(s) are selected soas to allow desired placement of an agent to allow conjugation to occurat a desired site. The reducing agent is, for example, TCEP. Thereduction reaction conditions such as, for example, the ratio ofreducing agent to activatable antibody, the length of incubation, thetemperature during the incubation, the pH of the reducing reactionsolution, etc., are determined by identifying the conditions thatproduce a conjugated activatable antibody in which the MM retains theability to effectively and efficiently mask the AB of the activatableantibody in an uncleaved state. The ratio of reduction agent toactivatable antibody will vary depending on the activatable antibody. Insome embodiments, the ratio of reducing agent to activatable antibodywill be in a range from about 20:1 to 1:1, from about 10:1 to 1:1, fromabout 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, fromabout 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, fromabout 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, fromabout 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5,from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In someembodiments, the ratio is in a range of from about 5:1 to 1:1. In someembodiments, the ratio is in a range of from about 5:1 to 1.5:1. In someembodiments, the ratio is in a range of from about 4:1 to 1:1. In someembodiments, the ratio is in a range from about 4:1 to 1.5:1. In someembodiments, the ratio is in a range from about 8:1 to about 1:1. Insome embodiments, the ratio is in a range of from about 2.5:1 to 1:1.

In some embodiments, a method of reducing interchain disulfide bonds inthe AB of an activatable antibody and conjugating an agent, e.g., athiol-containing agent such as a drug, to the resulting interchainthiols to selectively locate agent(s) on the AB is provided. The methodgenerally includes partially reducing the AB with a reducing agent toform at least two interchain thiols without forming all possibleinterchain thiols in the activatable antibody; and conjugating the agentto the interchain thiols of the partially reduced AB. For example, theAB of the activatable antibody is partially reduced for about 1 hour atabout 37° C. at a desired ratio of reducing agent:activatable antibody.In some embodiments, the ratio of reducing agent to activatable antibodywill be in a range from about 20:1 to 1:1, from about 10:1 to 1:1, fromabout 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, fromabout 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, fromabout 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, fromabout 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5,from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In someembodiments, the ratio is in a range of from about 5:1 to 1:1. In someembodiments, the ratio is in a range of from about 5:1 to 1.5:1. In someembodiments, the ratio is in a range of from about 4:1 to 1:1. In someembodiments, the ratio is in a range from about 4:1 to 1.5:1. In someembodiments, the ratio is in a range from about 8:1 to about 1:1. Insome embodiments, the ratio is in a range of from about 2.5:1 to 1:1.

The thiol-containing reagent can be, for example, cysteine or N-acetylcysteine. The reducing agent can be, for example, TCEP. In someembodiments, the reduced activatable antibody can be purified prior toconjugation, using for example, column chromatography, dialysis, ordiafiltration. In some embodiments, the reduced antibody is not purifiedafter partial reduction and prior to conjugation.

The disclosure also provides partially reduced activatable antibodies inwhich at least one interchain disulfide bond in the activatable antibodyhas been reduced with a reducing agent without disturbing any intrachaindisulfide bonds in the activatable antibody, wherein the activatableantibody includes an antibody or an antigen binding fragment thereof(AB) that specifically binds to the target, a masking moiety (MM) thatinhibits the binding of the AB of the activatable antibody in anuncleaved state to the target, and a cleavable moiety (CM) coupled tothe AB, wherein the CM is a polypeptide that functions as a substratefor at least one MMP. In some embodiments, the MM is coupled to the ABvia the CM. In some embodiments, one or more intrachain disulfidebond(s) of the activatable antibody is not disturbed by the reducingagent. In some embodiments, one or more intrachain disulfide bond(s) ofthe MM within the activatable antibody is not disturbed by the reducingagent. In some embodiments, the activatable antibody in the uncleavedstate has the structural arrangement from N-terminus to C-terminus asfollows: MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent isTCEP.

The disclosure also provides partially reduced activatable antibodies,including but not limited to multispecific activatable antibodies of thedisclosure, in which at least one interchain disulfide bond in theactivatable antibody has been reduced with a reducing agent withoutdisturbing or otherwise compromising the activity and/or efficacy of theactivatable antibody, wherein the activatable antibody includes anantibody or an antigen binding fragment thereof (AB) that specificallybinds to a target, a masking moiety (MM) that inhibits the binding ofthe AB of the activatable antibody in an uncleaved state to the target,and a cleavable moiety (CM) coupled to the AB, and the CM is apolypeptide that functions as a substrate for a protease. The activityand/or efficacy of the activatable antibody is, by way of nonlimitingexample, masking activity, activation of the activatable antibody,and/or binding activity of the activated activatable antibody. In someembodiments, one or more intrachain disulfide bond(s) of the activatableantibody is not disturbed by the reducing agent. In some embodiments,one or more intrachain disulfide bond(s) of the MM within theactivatable antibody is not disturbed by the reducing agent. In someembodiments, the activatable antibody in the uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP.

The disclosure also provides conjugated activatable antibodies thatinclude an activatable antibody linked to monomethyl auristatin D (MMAD)payload, wherein the activatable antibody includes an antibody or anantigen binding fragment thereof (AB) that specifically binds to atarget, a masking moiety (MM) that inhibits the binding of the AB of theactivatable antibody in an uncleaved state to the target, and cleavablemoiety (CM) coupled to the AB, and the CM is a polypeptide thatfunctions as a substrate for at least one MMP protease.

In some embodiments, the MMAD-conjugated activatable antibody can beconjugated using any of several methods for attaching agents to ABs: (a)attachment to the carbohydrate moieties of the AB, or (b) attachment tosulfhydryl groups of the AB, or (c) attachment to amino groups of theAB, or (d) attachment to carboxylate groups of the AB.

In some embodiments, the MMAD payload is conjugated to the AB via alinker. In some embodiments, the MMAD payload is conjugated to acysteine in the AB via a linker. In some embodiments, the MMAD payloadis conjugated to a lysine in the AB via a linker. In some embodiments,the MMAD payload is conjugated to another residue of the AB via alinker, such as those residues disclosed herein. In some embodiments,the linker is a thiol-containing linker. In some embodiments, the linkeris a cleavable linker. In some embodiments, the linker is anon-cleavable linker. In some embodiments, the linker is selected fromthe group consisting of the linkers shown in Tables 5 and 6. In someembodiments, the activatable antibody and the MMAD payload are linkedvia a maleimide caproyl-valine-citrulline linker. In some embodiments,the activatable antibody and the MMAD payload are linked via a maleimidePEG-valine-citrulline linker. In some embodiments, the activatableantibody and the MMAD payload are linked via a maleimidecaproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In someembodiments, the activatable antibody and the MMAD payload are linkedvia a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyllinker. In some embodiments, the MMAD payload is conjugated to the ABusing the partial reduction and conjugation technology disclosed herein.

In some embodiments, the target is selected from the group of targetslisted in Table 1. In some embodiments, the target is EGFR. In someembodiments, the target is a Jagged protein, e.g., Jagged 1 and/orJagged 2. In some embodiments, the target is interleukin 6 receptor(IL-6R). In some embodiments, the AB is or is derived from an antibodyselected from the group of antibodies listed in Table 2. In someembodiments, the antigen binding fragment thereof is selected from thegroup consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb,a dAb, a single domain heavy chain antibody, and a single domain lightchain antibody. In some embodiments, the AB has an equilibriumdissociation constant of about 100 nM or less for binding to the target.In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target. In some embodiments, the MM does notinterfere or compete with the AB of the activatable antibody in acleaved state for binding to the target. In some embodiments, the MM isa polypeptide of no more than 40 amino acids in length. In someembodiments, the MM polypeptide sequence is different from that of thetarget, and the MM polypeptide sequence is no more than 50% identical toany natural binding partner of the AB. In some embodiments, the MM doesnot include more than 25% amino acid sequence identity to the target. Insome embodiments, the MM does not include more than 10% amino acidsequence identity to the target. In some embodiments, the CM is apolypeptide of up to 15 amino acids in length. In some embodiments, theMMP protease is co-localized with the target in a tissue, and the MMPprotease cleaves the CM in the activatable antibody when the activatableantibody is exposed to the MMP protease. In some embodiments, the MMPprotease is a MMP9 protease. In some embodiments, the MMP protease is aMMP14 protease. In some embodiments, the activatable antibody includes alinking peptide between the MM and the CM. In some embodiments, theactivatable antibody includes a linking peptide between the CM and theAB. In some embodiments, the activatable antibody includes a firstlinking peptide (LP1) and a second linking peptide (LP2), and theactivatable antibody in an uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-ABor AB-LP2-CM-LP 1-MM. In some embodiments, the two linking peptides neednot be identical to each other. In some embodiments, each of LP1 and LP2is a peptide of about 1 to 20 amino acids in length. In someembodiments, at least one of LP1 or LP2 includes an amino acid sequenceselected from the group consisting of (GS)_(n), (GGS)_(n), (GSGGS)_(n)(SEQ ID NO: 1) and (GGGS)_(n) (SEQ ID NO: 2), where n is an integer ofat least one. In some embodiments, at least one of LP1 or LP2 includesan amino acid sequence selected from the group consisting of GGSG (SEQID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8). In some embodiments,the activatable antibody includes a second CM; in some embodiments, thesecond CM is a substrate for an enzyme selected from the groupconsisting of those shown in Table 7.

The disclosure also provides polypeptides and other larger moleculesthat include one or more of the MMP-cleavable substrate sequencespresented herein. By way of non-limiting example, the MMP-cleavablesubstrate sequences presented herein are useful in prodrug compositionsand methods of use thereof. These MMP-cleavable substrate sequencespresented herein are also useful in probes and other detection agentsand methods of use thereof. For example, the MMP-cleavable substratesequences presented herein can be used in conjunction with fluors andother quenchers to produce detection agents, such as imaging agentsand/or other diagnostic agents. Those of ordinary skill in the art willappreciate that the MMP-cleavable substrate sequences presented hereinare useful in any composition and/or method in the art that would use asubstrate that is cleavable by one or more MMPs, such as MMP9 and/orMMP14.

The disclosure also provides an isolated nucleic acid molecule encodingan antibody and/or an activatable antibody described herein, as well asvectors that include these isolated nucleic acid sequences. Thedisclosure provides methods of producing an antibody and/or activatableantibody by culturing a cell under conditions that lead to expression ofthe antibody and/or activatable antibody, wherein the cell comprisessuch a vector.

The disclosure provides a method of manufacturing a conjugated antibodyof the disclosure that bind a given target by (a) culturing a cellcomprising a nucleic acid construct that encodes the antibody underconditions that lead to expression of the antibody, (i) wherein theantibody includes a cleavable moiety (CM), and (ii) wherein the CM is apolypeptide that functions as a substrate for a matrix metalloprotease;(b) recovering the antibody; and (c) conjugating the recovered antibodyto one or more additional agents.

The disclosure also provides a method of manufacturing the activatableantibodies of the disclosure that bind in an activated state a giventarget by (a) culturing a cell comprising a nucleic acid construct thatencodes the activatable antibody under conditions that lead toexpression of the activatable antibody, wherein the activatable antibodycomprises a masking moiety (MM), a cleavable moiety (CM), and anantibody or an antigen binding fragment thereof (AB) that specificallybinds the target, (i) wherein the CM is a polypeptide that functions asa substrate for a MMP; and (ii) wherein the CM is positioned in theactivatable antibody such that, in an uncleaved state, the MM interfereswith specific binding of the AB to the target and in a cleaved state theMM does not interfere or compete with specific binding of the AB to thetarget; and (b) recovering the activatable antibody.

The disclosure provides methods of preventing, delaying the progressionof, treating, alleviating a symptom of, or otherwise ameliorating atarget-related disease in a subject by administering a therapeuticallyeffective amount of a conjugated antibody, an activatable antibodyand/or a conjugated activatable antibody described herein to a subjectin need thereof.

The disclosure provides methods of preventing, delaying the progressionof, treating, alleviating a symptom of, or otherwise amelioratinginflammation and/or an inflammatory disorder in a subject byadministering a therapeutically effective amount of a conjugatedantibody, an activatable antibody and/or a conjugated activatableantibody described herein to a subject in need thereof. The disclosurealso provides methods of preventing, delaying the progression of,treating, alleviating a symptom of, or otherwise ameliorating cancer ina subject by administering a therapeutically effective amount of aconjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody described herein to a subject in need thereof. Thedisclosure also provides methods of preventing, delaying the progressionof, treating, alleviating a symptom of, or otherwise ameliorating anautoimmune disease in a subject by administering a therapeuticallyeffective amount a conjugated antibody, an activatable antibody and/or aconjugated activatable antibody described herein to a subject in needthereof.

A conjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody used in any of the embodiments of these methods anduses can be administered at any stage of the disease. For example, sucha conjugated antibody, activatable antibody and/or conjugatedactivatable antibody can be administered to a patient suffering cancerof any stage, from early to metastatic. The terms subject and patientare used interchangeably herein.

In some embodiments, the subject is a mammal, such as a human, non-humanprimate, companion animal (e.g., cat, dog, horse), farm animal, workanimal, or zoo animal. In some embodiments, the subject is a rodent. Insome embodiments, the subject is a human. In some embodiments, thesubject is a companion animal. In some embodiments, the subject is ananimal in the care of a veterinarian.

The conjugated antibody, activatable antibody and/or conjugatedactivatable antibody and therapeutic formulations thereof areadministered to a subject suffering from or susceptible to a disease ordisorder associated with aberrant target expression and/or activity. Asubject suffering from or susceptible to a disease or disorderassociated with aberrant target expression and/or activity is identifiedusing any of a variety of methods known in the art. For example,subjects suffering from cancer or other neoplastic condition areidentified using any of a variety of clinical and/or laboratory testssuch as, physical examination and blood, urine and/or stool analysis toevaluate health status. For example, subjects suffering frominflammation and/or an inflammatory disorder are identified using any ofa variety of clinical and/or laboratory tests such as physicalexamination and/or bodily fluid analysis, e.g., blood, urine and/orstool analysis, to evaluate health status.

Administration of a conjugated antibody, an activatable antibody and/ora conjugated activatable antibody to a patient suffering from a diseaseor disorder associated with aberrant target expression and/or activityis considered successful if any of a variety of laboratory or clinicalobjectives is achieved. For example, administration of a conjugatedantibody, an activatable antibody and/or a conjugated activatableantibody to a patient suffering from a disease or disorder associatedwith aberrant target expression and/or activity is considered successfulif one or more of the symptoms associated with the disease or disorderis alleviated, reduced, inhibited or does not progress to a further,i.e., worse, state. Administration of a conjugated antibody, anactivatable antibody and/or a conjugated activatable antibody to apatient suffering from a disease or disorder associated with aberranttarget expression and/or activity is considered successful if thedisease or disorder enters remission or does not progress to a further,i.e., worse, state.

In some embodiments, the conjugated antibody, activatable antibodyand/or conjugated activatable antibody is administered during and/orafter treatment in combination with one or more additional agents suchas, by way of non-limiting example, an anti-inflammatory agent, animmunosuppressive agent, a chemotherapeutic agent, such as an alkylatingagent, an anti-metabolite, an anti-microtubule agent, a topoisomeraseinhibitor, a cytotoxic antibiotic, and/or any other nucleic aciddamaging agent. In some embodiments, the additional agent is a taxane,such as paclitaxel (e.g., Abraxane®). In some embodiments, theadditional agent is an anti-metabolite, such as gemcitabine. In someembodiments, the additional agent is an alkylating agent, such asplatinum-based chemotherapy, such as carboplatin or cisplatin. In someembodiments, the additional agent is a targeted agent, such as a kinaseinhibitor, e.g., sorafenib or erlotinib. In some embodiments, theadditional agent is a targeted agent, such as another antibody, e.g., amonoclonal antibody (e.g., bevacizumab), a bispecific antibody, or amultispecific antibody. In some embodiments, the additional agent is aproteosome inhibitor, such as bortezomib or carfilzomib. In someembodiments, the additional agent is an immune modulating agent, such aslenolidominde or IL-2. In some embodiments, the additional agent isradiation. In some embodiments, the additional agent is an agentconsidered standard of care by those skilled in the art. In someembodiments, the additional agent is a chemotherapeutic agent well knownto those skilled in the art.

In some embodiments, the additional agent is an antibody, anotherconjugated antibody, another activatable antibody and/or anotherconjugated activatable antibody. In some embodiments, the conjugatedantibody, activatable antibody and/or conjugated activatable antibodyand the additional agent(s) are administered simultaneously. Forexample, the conjugated antibody, activatable antibody and/or conjugatedactivatable antibody and the additional agent(s) can be formulated in asingle composition or administered as two or more separate compositions.In some embodiments, the conjugated antibody, activatable antibodyand/or conjugated activatable antibody and the additional agent(s) areadministered sequentially, or the antibody and/or conjugated antibodiesand the additional agent are administered at different times during atreatment regimen. For example, the antibody and/or conjugatedantibodies is administered prior to the administration of the additionalagent, the antibody and/or conjugated antibodies is administeredsubsequent to the administration of the additional agent, or theantibody and/or conjugated antibodies and the additional agent areadministered in an alternating fashion. As described herein, theantibody and/or conjugated antibodies and additional agent areadministered in single doses or in multiple doses.

In some embodiments, the CM is linked or otherwise attached to anactivatable antibody that includes an antibody or antigen-bindingfragment thereof that specifically binds a given target coupled to amasking moiety (MM), such that coupling of the MM to the AB reduces theability of the antibody or antigen-binding fragment thereof to bind thetarget. In some embodiments, the MM is coupled via the CM. Exemplarytargets include, but are not limited to the targets shown in Table 1.Exemplary ABs include, but are not limited to, the targets shown inTable 2. The activatable antibodies provided herein are stable incirculation, activated at intended sites of therapy and/or diagnosis butnot in normal, e.g., healthy tissue or other tissue not targeted fortreatment and/or diagnosis, and, when activated, exhibit binding to thetarget that is at least comparable to the corresponding, unmodifiedantibody.

The disclosure also provides methods and kits for using the conjugatedantibodies, activatable antibodies and/or conjugated activatableantibodies in a variety of diagnostic and/or prophylactic indications.

In some embodiments, the disclosure provides methods and kits fordetecting presence or absence of a cleaving agent and a target ofinterest in a subject or a sample by (i) contacting a subject or samplewith an activatable antibody, wherein the activatable antibody comprisesa masking moiety (MM), a cleavable moiety (CM) that is cleaved by thecleaving agent, and an antigen binding domain or fragment thereof (AB)that specifically binds the target of interest, wherein the activatableantibody in an uncleaved, non-activated state comprises a structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of theAB to the target, and wherein the MM does not have an amino acidsequence of a naturally occurring binding partner of the AB and is not amodified form of a natural binding partner of the AB; and (b) wherein,in an uncleaved, non-activated state, the MM interferes with specificbinding of the AB to the target, and in a cleaved, activated state theMM does not interfere or compete with specific binding of the AB to thetarget; and (ii) measuring a level of activated activatable antibody inthe subject or sample, wherein a detectable level of activatedactivatable antibody in the subject or sample indicates that thecleaving agent and the target are present in the subject or sample andwherein no detectable level of activated activatable antibody in thesubject or sample indicates that the cleaving agent, the target or boththe cleaving agent and the target are absent in the subject or sample.

In some embodiments, the activatable antibody is an activatable antibodyto which a therapeutic agent is conjugated. In some embodiments, theactivatable antibody is not conjugated to an agent. In some embodiments,the activatable antibody comprises a detectable label. In someembodiments, the detectable label is positioned on the AB. In someembodiments, measuring the level of activatable antibody in the subjector sample is accomplished using a secondary reagent that specificallybinds to the activated antibody, wherein the reagent comprises adetectable label. In some embodiments, the secondary reagent is anantibody comprising a detectable label.

In some embodiments of these methods and kits, the activatable antibodyincludes a detectable label. In some embodiments of these methods andkits, the detectable label includes an imaging agent, a contrastingagent, an enzyme, a fluorescent label, a chromophore, a dye, one or moremetal ions, or a ligand-based label. In some embodiments of thesemethods and kits, the imaging agent comprises a radioisotope. In someembodiments of these methods and kits, the radioisotope is indium ortechnetium. In some embodiments of these methods and kits, thecontrasting agent comprises iodine, gadolinium or iron oxide. In someembodiments of these methods and kits, the enzyme comprises horseradishperoxidase, alkaline phosphatase, or β-galactosidase. In someembodiments of these methods and kits, the fluorescent label comprisesyellow fluorescent protein (YFP), cyan fluorescent protein (CFP), greenfluorescent protein (GFP), modified red fluorescent protein (mRFP), redfluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europiumderivative. In some embodiments of these methods and kits, theluminescent label comprises an N-methylacrydium derivative. In someembodiments of these methods, the label comprises an Alexa Fluor® label,such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments ofthese methods and kits, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

In some embodiments of these methods and kits, the subject is a mammal.In some embodiments of these methods, the subject is a human. In someembodiments, the subject is a non-human mammal, such as a non-humanprimate, companion animal (e.g., cat, dog, horse), farm animal, workanimal, or zoo animal. In some embodiments, the subject is a rodent. Insome embodiments, the subject is a human. In some embodiments, thesubject is a companion animal. In some embodiments, the subject is ananimal in the care of a veterinarian.

In some embodiments of these methods and kits, the method is an in vivomethod. In some embodiments of these methods, the method is an in situmethod. In some embodiments of these methods, the method is an ex vivomethod. In some embodiments of these methods, the method is an in vitromethod.

In some embodiments of the methods and kits, the method is used toidentify or otherwise refine a patient population suitable for treatmentwith an activatable antibody of the disclosure, followed by treatment byadministering that activatable antibody and/or conjugated activatableantibody to a subject in need thereof. For example, patients that testpositive for both the target and at least one MMP that cleaves thesubstrate in the cleavable moiety (CM) of the activatable antibody beingtested in these methods are identified as suitable candidates fortreatment with such an activatable antibody comprising such a CM, andthe patient is then administered a therapeutically effective amount ofthe activatable antibody and/or conjugated activatable antibody that wastested. Likewise, patients that test negative for either or both of thetarget and the MMP that cleaves the substrate in the CM in theactivatable antibody being tested using these methods might beidentified as suitable candidates for another form of therapy. In someembodiments, such patients can be tested with other activatableantibodies until a suitable activatable antibody for treatment isidentified (e.g., an activatable antibody comprising a CM that iscleaved by the patient at the site of disease). In some embodiments, thepatient is then administered a therapeutically effective amount of theactivatable antibody and/or conjugated for which the patient testedpositive.

Pharmaceutical compositions according to the disclosure can include anantibody of the disclosure and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a series of graphs depicting the ability of theactivatable anti-EGFR antibody containing a masking moiety comprisingamino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 160), a cleavage moietycomprising the MMP14 substrate 520 (also referred to herein as MN520)ISSGLLSS (SEQ ID NO: 14), and the heavy and light chains of theanti-EGFR antibody C225v5, where the entire activatable antibodyconstruct is referred to herein as Pb-MN520, to inhibit tumor growth inthe H292 xenograft lung cancer model.

FIGS. 2A and 2B are a series of graphs depicting cleavage of thesubstrate pool referred to herein as SMP87 by 5 nM MMP9.

FIGS. 3A and 3B are a series of graphs depicting cleavage of substratesequence VAGRSMRP (SEQ ID NO: 484) by 5 nM MMP9.

FIG. 4 is a graph depicting correlation of substrate sequence frequencyand function.

FIGS. 5A and 5B are a series of graphs depicting cleavage of thesubstrate pool SMP39 by 60 nM MMP14.

FIGS. 6A and 6B are a series of graphs depicting cleavage of thesubstrate sequence QNQALRMA (SEQ ID NO: 15) by 30 nM MMP14.

FIGS. 7A and 7B are a series of schematic representations of the peptidedisplay platforms used in the working examples provided herein. FIG. 7Ais a schematic representation of the sequence of the display platformreferred to herein as “Display Platform CYTX-DP-XXXXXXXX” or“CYTX-DP-XXXXXXXX” (SEQ ID NO: 512). FIG. 7B is a schematicrepresentation of the sequence of the display platform referred toherein as “Display Platform SP-CYTX-DP-XXXXXXXX” or“SP-CYTX-DP-XXXXXXXX” (SEQ ID NO: 513), where SP-CYTX-DP-XXXXXXXX is theCYTX-DP-XXXXXXXX platform with a signal peptide.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure provides amino acid sequences that include a cleavablemoiety (CM) that is a substrate for at least one matrix metalloprotease(MMP). These CMs are useful in a variety of therapeutic, diagnostic andprophylactic indications.

The working examples provided herein demonstrate that these CM, whendisplayed in a peptide display platform, exhibit a number of desirablecleavage characteristics when exposed to an MMP protease under specifiedconditions. For example, Table 9 depicts (a) the percentage ofMMP9-selected substrates tested in the CYTX-DP display platform thatexhibited at least 20% cleavage when incubated with 50 nM human MMP9 for1 hour at 37° C. in 50 mM Tris-HCl, pH 7.4, supplemented with 150 mMNaCl, 10 mM CaCl₂, and 0.05% (w/v) Brij-35 (>20% Cleavage with 50 nMMMP9); (b) the percentage of MMP14-selected substrates tested in theCYTX-DP display platform that exhibited at least 20% cleavage whenincubated with 50 nM human MMP14 for 1 hour at 37° C. in 50 mM HEPES, pH6.8, supplemented with 10 mM CaCl₂, and 0.5 mM MgCl₂ (>20% Cleavage with50 nM MMP14); and (c) the percentage of MMP9-selected or MMP-14-selectedsubstrates tested in the CYTX-DP display platform that exhibited lessthan 20% cleavage when incubated with 500 pM human plasmin for 1 hour at37° C. in 50 mM Tris-HCl, pH 7.4, supplemented with 100 mM NaCl, 0.01%Tween20 and 1 mM EDTA (<20% cleavage with 500 pM plasmin).

In some embodiments, a MMP9 substrate when displayed in the CYTX-DPplatform exhibits at least 20% cleavage when incubated with 50 nM humanMMP9 for 1 hour at 37° C. in 50 mM Tris-HCl, pH 7.4, supplemented with150 mM NaCl, 10 mM CaCl₂, and 0.05% (w/v) Brij-35. In some embodiments,a MMP9 substrate when displayed in the CYTX-DP platform exhibits lessthan 20% cleavage when incubated with 500 pM human plasmin for 1 hour at37° C. in 50 mM Tris-HCl, pH 7.4, supplemented with 100 mM NaCl, 0.01%Tween20 and 1 mM EDTA. In some embodiments, a MMP9 substrate whendisplayed in the CYTX-DP platform exhibits at least 20% cleavage whenincubated with 50 nM human MMP9 for 1 hour at 37° C. in 50 mM Tris-HCl,pH 7.4, supplemented with 150 mM NaCl, 10 mM CaCl₂, and 0.05% (w/v)Brij-35 and exhibits less than 20% cleavage when incubated with 500 pMhuman plasmin for 1 hour at 37° C. in 50 mM Tris-HCl, pH 7.4,supplemented with 100 mM NaCl, 0.01% Tween20 and 1 mM EDTA.

In some embodiments a MMP14 substrate exhibits at least 20% cleavagewhen incubated with 50 nM human MMP14 for 1 hour at 37° C. in 50 mMHEPES, pH 6.8, supplemented with 10 mM CaCl₂, and 0.5 mM MgCl₂. In someembodiments, a MMP14 substrate when displayed in the CYTX-DP platformexhibits less than 20% cleavage when incubated with 500 pM human plasminfor 1 hour at 37° C. in 50 mM Tris-HCl, pH 7.4, supplemented with 100 mMNaCl, 0.01% Tween20 and 1 mM EDTA. In some embodiments a MMP14 substrateexhibits at least 20% cleavage when incubated with 50 nM human MMP14 for1 hour at 37° C. in 50 mM HEPES, pH 6.8, supplemented with 10 mM CaCl₂,and 0.5 mM MgCl₂ and exhibits less than 20% cleavage when incubated with500 pM human plasmin for 1 hour at 37° C. in 50 mM Tris-HCl, pH 7.4,supplemented with 100 mM NaCl, 0.01% Tween20 and 1 mM EDTA.

In some embodiments, the observed k_(cat)/K_(M) value of a substrate inan activatable antibody for MMP9 is greater than 100 M⁻¹ s⁻¹. In someembodiments, the observed k_(cat)/K_(M) value of a substrate in anactivatable antibody for MMP9 is greater than 1,000 M⁻¹ s⁻¹. In someembodiments, the observed k_(cat)/K_(M) value of a substrate in anactivatable antibody for MMP9 is greater than 10,000 M⁻¹ s⁻¹.

In some embodiments, the observed k_(cat)/K_(M) value of a substrate inan activatable antibody for MMP14 is greater than 100 M⁻¹ s⁻¹. In someembodiments, the observed k_(cat)/K_(M) value of a substrate in anactivatable antibody for MMP14 is greater than 1,000 M⁻¹ s⁻¹. In someembodiments, the observed k_(cat)/K_(M) value of a substrate in anactivatable antibody for MMP14 is greater than 10,000 M⁻¹ s⁻¹.

The disclosure also provides antibodies that include one or more ofthese MMP-cleavable substrates. For example, these MMP-cleavablesubstrates are useful when conjugating antibodies to one or moreadditional agents to produce conjugated antibodies. These MMP-cleavableare useful in activatable antibody constructs.

The conjugated antibodies and/or activatable antibodies include anantibody or antigen-binding fragment thereof (AB) that specificallybinds a target. Exemplary classes of targets of an AB include, but arenot necessarily limited to, cell surface receptors and secreted bindingproteins (e.g., growth factors), soluble enzymes, structural proteins(e.g. collagen, fibronectin) and the like. In some embodiments,conjugated antibodies and/or activatable antibodies have an AB thatbinds an extracellular target, usually an extracellular protein target.In some embodiments, conjugated antibodies and/or activatable antibodiesare designed for cellular uptake and are switchable inside a cell.

As a non-limiting example, the AB is a binding partner for any targetlisted in Table 1.

TABLE 1 Exemplary Targets 1-92-LFA-3 CD52 DL44 HVEM LIF-R STEAP1 Alpha-4CD56 DLK1 Hyaluronidase Lewis X STEAP2 integrin Alpha-V CD64 DLL4 ICOSLIGHT TAG-72 integrin alpha4beta1 CD70 DPP-4 IFNalpha LRP4 TAPA1integrin alpha4beta7 CD71 DSG1 IFNbeta LRRC26 TGFbeta integrin AGR2 CD74EGFR IFNgamma MCSP TIGIT Anti-Lewis-Y EGFRviii IgE Mesothelin TIM-3Apelin J CD80 Endothelin B IgE Receptor MRP4 TLR2 receptor receptor(FceRI) (ETBR) APRIL CD81 ENPP3 IGF MUC1 TLR4 B7-H4 CD86 EpCAM IGF1RMucin-16 TLR6 (MUC16, CA-125) BAFF CD95 EPHA2 IL1B Na/K ATPase TLR7 BTLACD117 EPHB2 IL1R Neutrophil TLR8 elastase C5 CD125 ERBB3 IL2 NGF TLR9complement C-242 CD132 F protein of IL11 Nicastrin TMEM31 (IL-2RG) RSVCA9 CD133 FAP IL12 Notch TNFalpha Receptors CA19-9 CD137 FGF-2 IL12p40Notch 1 TNFR (Lewis a) Carbonic CD138 FGF8 IL-12R, Notch 2 TNFRS12Aanhydrase 9 IL-12Rbeta1 CD2 CD166 FGFR1 IL13 Notch 3 TRAIL-R1 CD3 CD172AFGFR2 IL13R Notch 4 TRAIL-R2 CD6 CD248 FGFR3 IL15 NOV Transferrin CD9CDH6 FGFR4 IL17 OSM-R Transferrin receptor CD11a CEACAM5 Folate IL18OX-40 TRK-A (CEA) receptor CD19 CEACAM6 GAL3ST1 IL21 PAR2 TRK-B (NCA-90)CD20 CLAUDIN-3 G-CSF IL23 PDGF-AA uPAR CD22 CLAUDIN-4 G-CSFR IL23RPDGF-BB VAP1 CD24 cMet GD2 IL27/IL27R PDGFRalpha VCAM-1 (wsx1) CD25Collagen GITR IL29 PDGFRbeta VEGF CD27 Cripto GLUT1 IL-31R PD-1 VEGF-ACD28 CSFR GLUT4 IL31/IL31R PD-L1 VEGF-B CD30 CSFR-1 GM-CSF IL2R PD-L2VEGF-C CD33 CTLA-4 GM-CSFR IL4 Phosphatidyl- VEGF-D serine CD38 CTGF GPIIb/IIIa IL4R P1GF VEGFR1 receptors CD40 CXCL10 Gp130 IL6, IL6R PSCAVEGFR2 CD40L CXCL13 GPIIB/IIIA Insulin PSMA VEGFR3 Receptor CD41 CXCR1GPNMB Jagged RAAG12 VISTA Ligands CD44 CXCR2 GRP78 Jagged 1 RAGE WISP-1CD44v6 HER2/neu Jagged 2 SLC44A4 WISP-2 CD47 CXCR4 HGF LAG-3 Sphingosine1 WISP-3 Phosphate CD51 CYR61 hGH

As a non-limiting example, the AB is or is derived from an antibodylisted in Table 2.

TABLE 2 Exemplary sources for Abs Antibody Trade Name (antibody name)Target Avastin ™ (bevacizumab) VEGF Lucentis ™ (ranibizumab) VEGFErbitux ™ (cetuximab) EGFR Vectibix ™ (panitumumab) EGFR Remicade ™(infliximab) TNFα Humira ™ (adalimumab) TNFα Tysabri ™ (natalizumab)Integrinα4 Simulect ™ (basiliximab) IL2R Soliris ™ (eculizumab)Complement C5 Raptiva ™ (efalizumab) CD11a Bexxar ™ (tositumomab) CD20Zevalin ™ (ibritumomab tiuxetan) CD20 Rituxan ™ (rituximab) CD20Ocrelizumab CD20 Arzerra ™ (ofatumumab) CD20 Obinutuzumab CD20 Zenapax ™(daclizumab) CD25 Adcetris ™ (brentuximab vedotin) CD30 Myelotarg ™(gemtuzumab) CD33 Mylotarg ™ (gemtuzumab ozogamicin) CD33 Campath ™(alemtuzumab) CD52 ReoPro ™ (abiciximab) Glycoprotein receptor IIb/IIIaXolair ™ (omalizumab) IgE Herceptin ™ (trastuzumab) Her2 Kadcyla ™(trastuzumab emtansine) Her2 Synagis ™ (palivizumab) F protein of RSV(ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Hu5c8 CD40L (pertuzumab)Her2-neu (ertumaxomab) CD3/Her2-neu Orencia ™ (abatacept) CTLA-4(tanezumab) NGF (bavituximab) Phosphatidylserine (zalutumumab) EGFR(mapatumumab) EGFR (matuzumab) EGFR (nimotuzumab) EGFR ICR62 EGFR mAb528 EGFR CH806 EGFR MDX-447 EGFR/CD64 (edrecolomab) EpCAM RAV12 RAAG12huJ591 PSMA Enbrel ™ (etanercept) TNF-R Amevive ™ (alefacept) 1-92-LFA-3Antril ™, Kineret ™ (ankinra) IL-1Ra GC1008 TGFbeta Notch, e.g., Notch 1Jagged 1 or Jagged 2 (adecatumumab) EpCAM (figitumumab) IGF1R(tocilizumab) IL-6 receptor Stelara ™ (ustekinumab) IL-12/IL-23 Prolia ™(denosumab) RANKL

Exemplary conjugated antibodies and/or activatable antibodies of thedisclosure include, for example, antibodies that bind interleukin 6receptor (IL-6R) and that include a heavy chain and a light chain thatare, or are derived from, the antibody referred to herein as the“Av1”antibody, which binds interleukin-6 receptor (IL-6R). The amino acidsequences for the Av1 heavy chain and the Av1 light chain are shownbelow in SEQ ID NO: 54 and SEQ ID NO: 55, respectively.

Av1 Antibody Heavy Chain Amino Acid Sequence:

(SEQ ID NO: 54) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Av1 Antibody Light Chain Amino Acid Sequence:

(SEQ ID NO: 55) DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Exemplary conjugated antibodies and/or activatable antibodies of thedisclosure include, for example, antibodies that bind interleukin 6receptor (IL-6R) and that include a heavy chain and a light chain thatare, or are derived from, the Av1 antibody and a masking moiety.Exemplary conjugated antibodies and/or activatable antibodies of thedisclosure include an amino acid sequence attached to the N-terminus ofthe AV1 light chain. These N-terminal amino acid sequences include, forexample, YGSCSWNYVHIFMDC (SEQ ID NO: 161); QGDFDIPFPAHWVPIT (SEQ ID NO:162); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 163); QGQSGQYGSCSWNYVHIFMDC (SEQ IDNO: 164); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 165); orQGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 166). It is also to be appreciatedthat such amino acid sequences can be attached to the N-terminus of theAV1 heavy chain or to the C-terminus of the AV1 heavy or light chain.

Exemplary activatable antibodies of the disclosure include, for example,antibodies that bind Epidermal Growth Factor Receptor (EGFR) and thatinclude a heavy chain and a light chain that are, or are derived from,an antibody selected from the group consisting of the antibody referredto herein as the“c225v5” antibody, the antibody referred to herein asthe“c225v4” antibody, and the antibody referred to herein as the“c225v6”antibody, each of which binds EGFR. The c225v5 antibody, the c225v4antibody, and the c225v6 antibody share the same light chain sequence,referred to herein as “c225 light chain.” The amino acid sequences forthe c225v5 heavy chain, the c225v4 antibody, the c225v6 antibody, andthe c225 light chain are shown below.

C225v5 Antibody Heavy Chain Amino Acid Sequence:

(SEQ ID NO: 56) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

C225v4 Antibody Heavy Chain Amino Acid Sequence:

(SEQ ID NO: 57) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

C225v6 Antibody Heavy Chain Amino Acid Sequence:

(SEQ ID NO: 58) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

C225 Antibody Light Chain Amino Acid Sequence:

(SEQ ID NO: 59) QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC*

Exemplary conjugated antibodies and/or activatable antibodies of thedisclosure include, for example, antibodies that bind a Jagged target,e.g., Jagged-1, Jagged-2 and/or both Jagged-1 and Jagged-2, and thatinclude a combination of a variable heavy chain region and a variablelight chain region that are, or are derived from, the variable heavychain and variable light chain sequences shown below.

Variable Light Chain Amino Sequence Lc4 (SEQ ID NO: 60)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc4 (SEQ ID NO: 61)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc5 (SEQ ID NO: 62)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc5 (SEQ ID NO: 63)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYHGQFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc7 (SEQ ID NO: 64)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc7 (SEQ ID NO: 65)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc8 (SEQ ID NO: 67)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc8 (SEQ ID NO: 68)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHIGRTNPFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc13 (SEQ ID NO: 69)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc13 (SEQ ID NO: 70)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc16 (SEQ ID NO: 71)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc16 (SEQ ID NO: 72)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYYGQFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc19 (SEQ ID NO: 73)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc19 (SEQ ID NO: 74)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc21 (SEQ ID NO: 75)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc21 (SEQ ID NO: 76)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc24 (SEQ ID NO: 77)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc24 (SEQ ID NO: 78)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc26 (SEQ ID NO: 79)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc26 (SEQ ID NO: 80)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc27 (SEQ ID NO: 81)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc27 (SEQ ID NO: 82)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFYGQFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc28 (SEQ ID NO: 83)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc28 (SEQ ID NO: 84)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc30 (SEQ ID NO: 85)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc30 (SEQ ID NO: 86)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYAKSAAAFDYWGQGTLVTVSS Variable Light ChainAmino Sequence Lc31 (SEQ ID NO: 87)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc31 (SEQ ID NO: 88)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc32 (SEQ ID NO: 89)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc32 (SEQ ID NO: 90)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc37 (SEQ ID NO: 91)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc37 (SEQ ID NO: 92)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPHNGQFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc39 (SEQ ID NO: 93)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc39 (SEQ ID NO: 94)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc40 (SEQ ID NO: 95)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Heavy Chain Amino SequenceHc40 (SEQ ID NO: 96)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS Variable LightChain Amino Sequence Lc47 (SEQ ID NO: 97)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain AminoSequence Hc47 (SEQ ID NO: 98)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable 4B2 LightChain (SEQ ID NO: 99)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLDAPPQFGQGTKVEIKR Variable 4B2 Heavy Chain(SEQ ID NO: 100)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable 4D11Light Chain (SEQ ID NO: 101)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKR Variable 4D11 Heavy Chain(SEQ ID NO: 102)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable 4E7Light Chain (SEQ ID NO: 103)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLVAPLTFGQGTKVEIKR Variable 4E7 Heavy Chain(SEQ ID NO: 104)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable 4E11 LightChain (SEQ ID NO: 105)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALDAPLMFGQGTKVEIKR Variable 4E11 Heavy Chain(SEQ ID NO: 106)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEPMGQLTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable 6B7Light Chain (SEQ ID NO: 107)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR Variable 6B7 Heavy Chain(SEQ ID NO: 108)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable 6F8 LightChain (SEQ ID NO: 109)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR Variable 6F8 Heavy Chain(SEQ ID NO: 110)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS

Exemplary conjugated antibodies and/or activatable antibodies of thedisclosure include, for example, antibodies that bind a Jagged target,e.g., Jagged-1, Jagged-2 and/or both Jagged-1 and Jagged-2, and thatinclude a combination of a heavy chain region and a light chain regionthat are, or are derived from, the heavy chain and light chain sequencesshown below.

4D11 Light Chain sequence: (SEQ ID NO: 111)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 4D11Heavy Chain sequence: (SEQ ID NO: 112)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4D11v2 Heavy Chainsequence (SEQ ID NO: 113)EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4D11v2 Light ChainSequence (SEQ ID NO: 114)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLXKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

The activatable antibodies and activatable antibody compositionsprovided herein contain at least an antibody or antibody fragmentthereof (collectively referred to as AB throughout the disclosure) thatspecifically binds a target, e.g., a human target, wherein the AB ismodified by a masking moiety (MM).

In some embodiments, the masking moiety is selected for use with aspecific antibody or antibody fragment. For example, suitable maskingmoieties for use with antibodies that bind EGFR include MMs that includethe sequence CISPRG (SEQ ID NO: 167). By way of non-limiting examples,the MM can include a sequence such as CISPRGC (SEQ ID NO: 497); CISPRGCG(SEQ ID NO: 168); CISPRGCPDGPYVMY (SEQ ID NO: 160); CISPRGCPDGPYVM (SEQID NO: 169), CISPRGCEPGTYVPT (SEQ ID NO: 170) and CISPRGCPGQIWHPP (SEQID NO: 171). Other suitable masking moieties include any of theEGFR-specific masks disclosed in PCT Publication No. WO 2010/081173,such as, by way of non-limiting example, GSHCLIPINMGAPSC (SEQ ID NO:172); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 173);CNHHYFYTCGCISPRGCPG (SEQ ID NO: 174); ADHVFWGSYGCISPRGCPG (SEQ ID NO:175); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 176); CPHFTTTSCGCISPRGCPG (SEQ IDNO: 177); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 178); CPHVSFGSCGCISPRGCPG (SEQID NO: 179); CPYYTLSYCGCISPRGCPG (SEQ ID NO: 180); CNHVYFGTCGCISPRGCPG(SEQ ID NO: 181); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 182);CHHFTLTTCGCISPRGCPG (SEQ ID NO: 183); YNPCATPMCCISPRGCPG (SEQ ID NO:184); CNHHYFYTCGCISPRGCG (SEQ ID NO: 185); CNHHYHYYCGCISPRGCG (SEQ IDNO: 186); CNHVYFGTCGCISPRGCG (SEQ ID NO: 187); CHHVYWGHCGCISPRGCG (SEQID NO: 188); CPHFTTTSCGCISPRGCG (SEQ ID NO: 189); CNHFTLTTCGCISPRGCG(SEQ ID NO: 190); CHHFTLTTCGCISPRGCG (SEQ ID NO: 191);CPYYTLSYCGCISPRGCG (SEQ ID NO: 192); CPHVSFGSCGCISPRGCG (SEQ ID NO:193); ADHVFWGSYGCISPRGCG (SEQ ID NO: 194); YNPCATPMCCISPRGCG (SEQ ID NO:195); CHHVYWGHCGCISPRGCG (SEQ ID NO: 196);C(N/P)H(HN/F)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H)CGCISPRGCG (SEQ ID NO:197); CISPRGCGQPIPSVK (SEQ ID NO: 198); CISPRGCTQPYHVSR (SEQ ID NO:199); and/or CISPRGCNAVSGLGS (SEQ ID NO: 200).

Suitable masking moieties for use with antibodies that bind a Jaggedtarget, e.g., Jagged 1 and/or Jagged 2, include, by way of non-limitingexample, masking moieties that include a sequence such asQGQSGQCNIWLVGGDCRGWQG (SEQ ID NO: 496); QGQSGQGQQQWCNIWINGGDCRGWNG (SEQID NO: 201); PWCMQRQDFLRCPQP (SEQ ID NO: 202); QLGLPAYMCTFECLR (SEQ IDNO: 203); CNLWVSGGDCGGLQG (SEQ ID NO: 204); SCSLWTSGSCLPHSP (SEQ ID NO:205); YCLQLPHYMQAMCGR (SEQ ID NO: 206); CFLYSCTDVSYWNNT (SEQ ID NO:207); PWCMQRQDYLRCPQP (SEQ ID NO: 208); CNLWISGGDCRGLAG (SEQ ID NO:209); CNLWVSGGDCRGVQG (SEQ ID NO: 210); CNLWVSGGDCRGLRG (SEQ ID NO:211); CNLWISGGDCRGLPG (SEQ ID NO: 212); CNLWVSGGDCRDAPW (SEQ ID NO:213); CNLWVSGGDCRDLLG (SEQ ID NO: 214); CNLWVSGGDCRGLQG (SEQ ID NO:215); CNLWLHGGDCRGWQG (SEQ ID NO: 216); CNIWLVGGDCRGWQG (SEQ ID NO:217); CTTWFCGGDCGVMRG (SEQ ID NO: 218); CNIWGPSVDCGALLG (SEQ ID NO:219); CNIWVNGGDCRSFEG (SEQ ID NO: 220); YCLNLPRYMQDMCWA (SEQ ID NO:221); YCLALPHYMQADCAR (SEQ ID NO: 222); CFLYSCGDVSYWGSA (SEQ ID NO:223); CYLYSCTDSAFWNNR (SEQ ID NO: 224); CYLYSCNDVSYWSNT (SEQ ID NO:225); CFLYSCTDVSYW (SEQ ID NO: 226); CFLYSCTDVAYWNSA (SEQ ID NO: 227);CFLYSCTDVSYWGDT (SEQ ID NO: 228); CFLYSCTDVSYWGNS (SEQ ID NO: 229);CFLYSCTDVAYWNNT (SEQ ID NO: 230); CFLYSCGDVSYWGNPGLS (SEQ ID NO: 231);CFLYSCTDVAYWSGL (SEQ ID NO: 232); CYLYSCTDGSYWNST (SEQ ID NO: 233);CFLYSCSDVSYWGNI (SEQ ID NO: 234); CFLYSCTDVAYW (SEQ ID NO: 235);CFLYSCTDVSYWGST (SEQ ID NO: 236); CFLYSCTDVAYWGDT (SEQ ID NO: 237);GCNIWLNGGDCRGWVDPLQG (SEQ ID NO: 238); GCNIWLVGGDCRGWIGDTNG (SEQ ID NO:239); GCNIWLVGGDCRGWIEDSNG (SEQ ID NO: 240); GCNIWANGGDCRGWIDNIDG (SEQID NO: 241); GCNIWLVGGDCRGWLGEAVG (SEQ ID NO: 242); GCNIWLVGGDCRGWLEEAVG(SEQ ID NO: 243); GGPALCNIWLNGGDCRGWSG (SEQ ID NO: 244);GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 245); GQQQWCNIWINGGDCRGWNG (SEQ ID NO:246); GKSEFCNIWLNGGDCRGWIG (SEQ ID NO: 247); GTPGGCNIWANGGDCRGWEG (SEQID NO: 248); GASQYCNLWINGGDCRGWRG (SEQ ID NO: 249); GCNIWLVGGDCRPWVEGG(SEQ ID NO: 250); GCNIWAVGGDCRPFVDGG (SEQ ID NO: 251);GCNIWLNGGDCRAWVDTG (SEQ ID NO: 252); GCNIWIVGGDCRPFINDG (SEQ ID NO:253); GCNIWLNGGDCRPVVFGG (SEQ ID NO: 254); GCNIWLSGGDCRMFMNEG (SEQ IDNO: 255); GCNIWVNGGDCRSFVYSG (SEQ ID NO: 256); GCNIWLNGGDCRGWEASG (SEQID NO: 257); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 258); GCNIWLNGGDCRTFVASG(SEQ ID NO: 259); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 260);GFLENCNIWLNGGDCRTG (SEQ ID NO: 261); GIYENCNIWLNGGDCRMG (SEQ ID NO:262); and/or GIPDNCNIWINGGDCRYG (SEQ ID NO: 263).

Suitable masking moieties for use with antibodies that bind aninterleukin 6 target, e.g., interleukin 6 receptor (IL-6R), include, byway of non-limiting example, masking moieties that include a sequencesuch as QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 264); QGQSGQGDFDIPFPAHWVPIT(SEQ ID NO: 265); QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 266);YRSCNWNYVSIFLDC (SEQ ID NO: 267); PGAFDIPFPAHWVPNT (SEQ ID NO: 268);ESSCVWNYVHIYMDC (SEQ ID NO: 269); YPGCKWNYDRIFLDC (SEQ ID NO: 270);YRTCSWNYVGIFLDC (SEQ ID NO: 271); YGSCSWNYVHIFMDC (SEQ ID NO: 161);YGSCSWNYVHIFLDC (SEQ ID NO: 272); YGSCNWNYVHIFLDC (SEQ ID NO: 273);YTSCNWNYVHIFMDC (SEQ ID NO: 274); YPGCKWNYDRIFLDC (SEQ ID NO: 275);WRSCNWNYAHIFLDC (SEQ ID NO: 276); WSNCHWNYVHIFLDC (SEQ ID NO: 277);DRSCTWNYVRISYDC (SEQ ID NO: 278); SGSCKWDYVHIFLDC (SEQ ID NO: 279);SRSCIWNYAHIHLDC (SEQ ID NO: 280); SMSCYWQYERIFLDC (SEQ ID NO: 281);YRSCNWNYVSIFLDC (SEQ ID NO: 282); SGSCKWDYVHIFLDC (SEQ ID NO: 283);YKSCHWDYVHIFLDC (SEQ ID NO: 284); YGSCTWNYVHIFMEC (SEQ ID NO: 285);FSSCNWNYVHIFLDC (SEQ ID NO: 286); WRSCNWNYAHIFLDC (SEQ ID NO: 287);YGSCQWNYVHIFLDC (SEQ ID NO: 288); YRSCNWNYVHIFLDC (SEQ ID NO: 289);NMSCHWDYVHIFLDC (SEQ ID NO: 290); FGPCTWNYARISWDC (SEQ ID NO: 291);XXsCXWXYvhIfXdC (SEQ ID NO: 292); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 163);RDTGGQCRWDYVHIFMDC (SEQ ID NO: 293); AGVPAGCTWNYVHIFMEC (SEQ ID NO:294); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 295); DGGPAGCSWNYVHIFMEC (SEQ IDNO: 296); AVGPAGCWWNYVHIFMEC (SEQ ID NO: 297); CTWNYVHIFMDCGEGEGP (SEQID NO: 298); GGVPEGCTWNYAHIFMEC (SEQ ID NO: 299); AEVPAGCWWNYVHIFMEC(SEQ ID NO: 300); AGVPAGCTWNYVHIFMEC (SEQ ID NO: 301);SGASGGCKWNYVHIFMDC (SEQ ID NO: 302); TPGCRWNYVHIFMECEAL (SEQ ID NO:303); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 304); PGAFDIPFPAHWVPNT (SEQ ID NO:305); RGACDIPFPAHWIPNT (SEQ ID NO: 306); QGDFDIPFPAHWVPIT (SEQ ID NO:162); XGafDIPFPAHWvPnT (SEQ ID NO: 307); RGDGNDSDIPFPAHWVPRT (SEQ ID NO:308); SGVGRDRDIPFPAHWVPRT (SEQ ID NO: 309); WAGGNDCDIPFPAHWIPNT (SEQ IDNO: 310); WGDGMDVDIPFPAHWVPVT (SEQ ID NO: 311); AGSGNDSDIPFPAHWVPRT (SEQID NO: 312); ESRSGYADIPFPAHWVPRT (SEQ ID NO: 313); and/orRECGRCGDIPFPAHWVPRT (SEQ ID NO: 314).

When the AB is modified with a MM and is in the presence of the target,specific binding of the AB to its target is reduced or inhibited, ascompared to the specific binding of the AB not modified with an MM orthe specific binding of the parental AB to the target.

The K_(d) of the AB modified with a MM towards the target is at least 5,10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000,500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, orbetween 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,100,000-1,000,000, or 100,000-10,000,000 times greater than the K_(d) ofthe AB not modified with an MM or of the parental AB towards the target.Conversely, the binding affinity of the AB modified with a MM towardsthe target is at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500,1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100,10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000,10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or100,000-10,000,000 times lower than the binding affinity of the AB notmodified with an MM or of the parental AB towards the target.

The dissociation constant (K_(d)) of the MM towards the AB is generallygreater than the K_(d) of the AB towards the target. The K_(d) of the MMtowards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000,2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 timesgreater than the K_(d) of the AB towards the target. Conversely, thebinding affinity of the MM towards the AB is generally lower than thebinding affinity of the AB towards the target. The binding affinity ofMM towards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000,2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times lowerthan the binding affinity of the AB towards the target.

When the AB is modified with a MM and is in the presence of the targetspecific binding of the AB to its target is reduced or inhibited, ascompared to the specific binding of the AB not modified with an MM orthe specific binding of the parental AB to the target. When compared tothe binding of the AB not modified with an MM or the binding of theparental AB to the target the AB's ability to bind the target whenmodified with an MM can be reduced by at least 50%, 60%, 70%, 80%, 90%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4,6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30,45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 months or more when measured in vivo or in an in vitro assay.

The MM inhibits the binding of the AB to the target. The MM binds theantigen binding domain of the AB and inhibits binding of the AB to thetarget. The MM can sterically inhibit the binding of the AB to thetarget. The MM can allosterically inhibit the binding of the AB to itstarget. In these embodiments when the AB is modified or coupled to a MMand in the presence of target there is no binding or substantially nobinding of the AB to the target, or no more than 0.001%, 0.01%, 0.1%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,or 50% binding of the AB to the target, as compared to the binding ofthe AB not modified with an MM, the parental AB, or the AB not coupledto an MM to the target, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48,60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer whenmeasured in vivo or in an in vitro assay.

When an AB is coupled to or modified by a MM, the MM ‘masks’ or reducesor otherwise inhibits the specific binding of the AB to the target. Whenan AB is coupled to or modified by a MM, such coupling or modificationcan effect a structural change that reduces or inhibits the ability ofthe AB to specifically bind its target.

An AB coupled to or modified with an MM can be represented by thefollowing formulae (in order from an amino (N) terminal region tocarboxyl (C) terminal region:

(MM)-(AB)

(AB)-(MM)

(MM)-L-(AB)

(AB)-L-(MM)

where MM is a masking moiety, the AB is an antibody or antibody fragmentthereof, and the L is a linker. In many embodiments, it may be desirableto insert one or more linkers, e.g., flexible linkers, into thecomposition so as to provide for flexibility.

In certain embodiments, the MM is not a natural binding partner of theAB. In some embodiments, the MM contains no or substantially no homologyto any natural binding partner of the AB. In some embodiments, the MM isno more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or 80% similar to any natural binding partner of the AB.In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to anynatural binding partner of the AB. In some embodiments, the MM is nomore than 25% identical to any natural binding partner of the AB. Insome embodiments, the MM is no more than 50% identical to any naturalbinding partner of the AB. In some embodiments, the MM is no more than20% identical to any natural binding partner of the AB. In someembodiments, the MM is no more than 10% identical to any natural bindingpartner of the AB.

In some embodiments, the activatable antibodies include an AB that ismodified by an MM and also includes one or more cleavable moieties (CM).Such activatable antibodies exhibit activatable/switchable binding, tothe AB's target. Activatable antibodies generally include an antibody orantibody fragment (AB), modified by or coupled to a masking moiety (MM)and a modifiable or cleavable moiety (CM). In some embodiments, the CMcontains an amino acid sequence that serves as a substrate for at leastone matrix metalloprotease of interest.

The elements of the activatable antibodies are arranged so that the MMand CM are positioned such that in a cleaved (or relatively active)state and in the presence of a target, the AB binds a target while in anuncleaved (or relatively inactive) state in the presence of the target,specific binding of the AB to its target is reduced or inhibited. Thespecific binding of the AB to its target can be reduced due to theinhibition or masking of the AB's ability to specifically bind itstarget by the MM.

The K_(d) of the AB modified with a MM and a CM towards the target is atleast 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000,50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000,10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000,100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000,1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000,10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 timesgreater than the K_(d) of the AB not modified with an MM and a CM or ofthe parental AB towards the target. Conversely, the binding affinity ofthe AB modified with a MM and a CM towards the target is at least 2, 3,4, 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000,50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000,10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000,100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000,1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000,10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lowerthan the binding affinity of the AB not modified with an MM and a CM orof the parental AB towards the target.

When the AB is modified with a MM and a CM and is in the presence of thetarget but not in the presence of a modifying agent (for example a MMP),specific binding of the AB to its target is reduced or inhibited, ascompared to the specific binding of the AB not modified with an MM and aCM or of the parental AB to the target. When compared to the binding ofthe parental AB or the binding of an AB not modified with an MM and a CMto its target, the AB's ability to bind the target when modified with anMM and a CM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8,12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours or 5, 10, 15, 30, 45,60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 months or longer when measured in vivo or in an in vitro assay.

As used herein, the term cleaved state refers to the condition of theactivatable antibodies following modification of the CM by at least onematrix metalloprotease. The term uncleaved state, as used herein, refersto the condition of the activatable antibodies in the absence ofcleavage of the CM by a MMP. As discussed above, the term “activatableantibodies” is used herein to refer to an activatable antibody in bothits uncleaved (native) state, as well as in its cleaved state. It willbe apparent to the ordinarily skilled artisan that in some embodiments,a cleaved activatable antibody may lack an MM due to cleavage of the CMby protease, resulting in release of at least the MM (e.g., where the MMis not joined to the activatable antibodies by a covalent bond (e.g., adisulfide bond between cysteine residues).

By activatable or switchable is meant that the activatable antibodyexhibits a first level of binding to a target when in a inhibited,masked or uncleaved state (i.e., a first conformation), and a secondlevel of binding to the target in the uninhibited, unmasked and/orcleaved state (i.e., a second conformation), where the second level oftarget binding is greater than the first level of binding. In general,the access of target to the AB of the activatable antibody is greater inthe presence of a cleaving agent capable of cleaving the CM than in theabsence of such a cleaving agent. Thus, when the activatable antibody isin the uncleaved state, the AB is inhibited from target binding and canbe masked from target binding (i.e., the first conformation is such theAB cannot bind the target), and in the cleaved state the AB is notinhibited or is unmasked to target binding.

The CM and AB of the activatable antibodies are selected so that the ABrepresents a binding moiety for a given target, and the CM represents asubstrate for a MMP that is co-localized with the target at a treatmentsite or diagnostic site in a subject. The activatable antibodiesdisclosed herein find particular use where, for example, a MMP capableof cleaving a site in the CM is present at relatively higher levels intarget-containing tissue of a treatment site or diagnostic site than intissue of non-treatment sites (for example in healthy tissue).

In some embodiments, activatable antibodies provide for reduced toxicityand/or adverse side effects that could otherwise result from binding ofthe AB at non-treatment sites if the AB were not masked or otherwiseinhibited from binding to the target.

In general, an activatable antibody can be designed by selecting an ABof interest and constructing the remainder of the activatable antibodyso that, when conformationally constrained, the MM provides for maskingof the AB or reduction of binding of the AB to its target. Structuraldesign criteria can be to be taken into account to provide for thisfunctional feature.

Activatable antibodies exhibiting a switchable phenotype of a desireddynamic range for target binding in an inhibited versus an uninhibitedconformation are provided. Dynamic range generally refers to a ratio of(a) a maximum detected level of a parameter under a first set ofconditions to (b) a minimum detected value of that parameter under asecond set of conditions. For example, in the context of an activatableantibody, the dynamic range refers to the ratio of (a) a maximumdetected level of target protein binding to an activatable antibody inthe presence of a MMP capable of cleaving the CM of the activatableantibodies to (b) a minimum detected level of target protein binding toan activatable antibody in the absence of the protease. The dynamicrange of an activatable antibody can be calculated as the ratio of theequilibrium dissociation constant of an activatable antibody cleavingagent (e.g., enzyme) treatment to the equilibrium dissociation constantof the activatable antibodies cleaving agent treatment. The greater thedynamic range of an activatable antibody, the better the switchablephenotype of the activatable antibody. Activatable antibodies havingrelatively higher dynamic range values (e.g., greater than 1) exhibitmore desirable switching phenotypes such that target protein binding bythe activatable antibodies occurs to a greater extent (e.g.,predominantly occurs) in the presence of a cleaving agent (e.g., enzyme)capable of cleaving the CM of the activatable antibodies than in theabsence of a cleaving agent.

Activatable antibodies can be provided in a variety of structuralconfigurations. Exemplary formulae for activatable antibodies areprovided below. It is specifically contemplated that the N- toC-terminal order of the AB, MM and CM may be reversed within anactivatable antibody. It is also specifically contemplated that the CMand MM may overlap in amino acid sequence, e.g., such that the CM iscontained within the MM.

For example, activatable antibodies can be represented by the followingformula (in order from an amino (N) terminal region to carboxyl (C)terminal region:

(MM)-(CM)-(AB)

(AB)-(CM)-(MM)

where MM is a masking moiety, CM is a cleavable moiety, and AB is anantibody or fragment thereof. It should be noted that although MM and CMare indicated as distinct components in the formulae above, in allexemplary embodiments (including formulae) disclosed herein it iscontemplated that the amino acid sequences of the MM and the CM couldoverlap, e.g., such that the CM is completely or partially containedwithin the MM. In addition, the formulae above provide for additionalamino acid sequences that may be positioned N-terminal or C-terminal tothe activatable antibodies elements.

In certain embodiments, the MM is not a natural binding partner of theAB. In some embodiments, the MM contains no or substantially no homologyto any natural binding partner of the AB. In some embodiments, the MM isno more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or 80% similar to any natural binding partner of the AB.In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to anynatural binding partner of the AB. In some embodiments, the MM is nomore than 50% identical to any natural binding partner of the AB. Insome embodiments, the MM is no more than 25% identical to any naturalbinding partner of the AB. In some embodiments, the MM is no more than20% identical to any natural binding partner of the AB. In someembodiments, the MM is no more than 10% identical to any natural bindingpartner of the AB.

In many embodiments, it may be desirable to insert one or more linkers,e.g., flexible linkers, into the activatable antibody construct so as toprovide for flexibility at one or more of the MM-CM junction, the CM-ABjunction, or both. For example, the AB, MM, and/or CM may not contain asufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Glyand Ser, particularly Gly) to provide the desired flexibility. As such,the switchable phenotype of such activatable antibody constructs maybenefit from introduction of one or more amino acids to provide for aflexible linker. In addition, as described below, where the activatableantibody is provided as a conformationally constrained construct, aflexible linker can be operably inserted to facilitate formation andmaintenance of a cyclic structure in the uncleaved activatable antibody.

For example, in certain embodiments, an activatable antibody comprisesone of the following formulae (where the formula below represent anamino acid sequence in either N- to C-terminal direction or C- toN-terminal direction):

(MM)-L1-(CM)-(AB)

(MM)-(CM)-L2-(AB)

(MM)-L1-(CM)-L2-(AB)

wherein MM, CM, and AB are as defined above; wherein L1 and L2 are eachindependently and optionally present or absent, are the same ordifferent flexible linkers that include at least 1 flexible amino acid(e.g., Gly). In addition, the formulae above provide for additionalamino acid sequences that may be positioned N-terminal or C-terminal tothe activatable antibodies elements. Examples include, but are notlimited to, targeting moieties (e.g., a ligand for a receptor of a cellpresent in a target tissue) and serum half-life extending moieties(e.g., polypeptides that bind serum proteins, such as immunoglobulin(e.g., IgG) or serum albumin (e.g., human serum albumin (HAS)).

The CM is specifically cleaved by at least one MMP at a rate of about0.001-1500×10⁴M⁻¹ S⁻¹ or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1,2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750,1000, 1250, or 1500×10⁴ M⁻¹ S⁻¹.

For specific cleavage by an enzyme, contact between the enzyme and CM ismade. When the activatable antibody comprising an AB coupled to a MM anda CM is in the presence of target and sufficient enzyme activity, the CMcan be cleaved. Sufficient enzyme activity can refer to the ability ofthe enzyme to make contact with the CM and effect cleavage. It canreadily be envisioned that an enzyme may be in the vicinity of the CMbut unable to cleave because of other cellular factors or proteinmodification of the enzyme.

Linkers suitable for use in compositions described herein are generallyones that provide flexibility of the modified AB or the activatableantibodies to facilitate the inhibition of the binding of the AB to thetarget. Such linkers are generally referred to as flexible linkers.Suitable linkers can be readily selected and can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 amino acids in length.

Exemplary flexible linkers include glycine polymers (G)n, glycine-serinepolymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 1) and(GGGS)n (SEQ ID NO: 2), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers known in the art. Glycine and glycine-serine polymers arerelatively unstructured, and therefore may be able to serve as a neutraltether between components. Glycine accesses significantly more phi-psispace than even alanine, and is much less restricted than residues withlonger side chains (see Scheraga, Rev. Computational Chem. 11173-142(1992)). Exemplary flexible linkers include, but are not limited toGly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 4),Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 5), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 6),Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 7), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 8),and the like. The ordinarily skilled artisan will recognize that designof an activatable antibodies can include linkers that are all orpartially flexible, such that the linker can include a flexible linkeras well as one or more portions that confer less flexible structure toprovide for a desired activatable antibodies structure.

In some embodiments, the activatable antibodies described herein alsoinclude an agent conjugated to the activatable antibody. In someembodiments, the conjugated agent is a therapeutic agent, such as ananti-inflammatory and/or an antineoplastic agent. In such embodiments,the agent is conjugated to a carbohydrate moiety of the activatableantibody, for example, in some embodiments, where the carbohydratemoiety is located outside the antigen-binding region of the antibody orantigen-binding fragment in the activatable antibody. In someembodiments, the agent is conjugated to a sulfhydryl group of theantibody or antigen-binding fragment in the activatable antibody.

In some embodiments, the agent is a cytotoxic agent such as a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

In some embodiments, the agent is a detectable moiety such as, forexample, a label or other marker. For example, the agent is or includesa radiolabeled amino acid, one or more biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods), one or more radioisotopes or radionuclides, oneor more fluorescent labels, one or more enzymatic labels, and/or one ormore chemiluminescent agents. In some embodiments, detectable moietiesare attached by spacer molecules.

The disclosure also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Suitablecytotoxic agents include, for example, dolastatins and derivativesthereof (e.g. auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). Forexample, the agent is monomethyl auristatin E (MMAE) or monomethylauristatin D (MMAD). In some embodiments, the agent is an agent selectedfrom the group listed in Table 3. In some embodiments, the agent is adolastatin. In some embodiments, the agent is an auristatin orderivative thereof. In some embodiments, the agent is auristatin E or aderivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid ormaytansinoid derivative. In some embodiments, the agent is DM1 or DM4.In some embodiments, the agent is a duocarmycin or derivative thereof.In some embodiments, the agent is a calicheamicin or derivative thereof.In some embodiments, the agent is a pyrrolobenzodiazepine.

In some embodiments, the agent is linked to the AB using a maleimidecaproyl-valine-citrulline linker or a maleimide PEG-valine-citrullinelinker. In some embodiments, the agent is linked to the AB using amaleimide caproyl-valine-citrulline linker. In some embodiments, theagent is linked to the AB using a maleimide PEG-valine-citrulline linkerIn some embodiments, the agent is monomethyl auristatin D (MMAD) linkedto the AB using a maleimidePEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, and thislinker payload construct is referred to herein as “vc-MMAD.” In someembodiments, the agent is monomethyl auristatin E (MMAE) linked to theAB using a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyllinker, and this linker payload construct is referred to herein as“vc-MMAE.” The structures of vc-MMAD and vc-MMAE are shown below:

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Table 3 lists some of the exemplary pharmaceutical agents that may beemployed in the herein described disclosure but in no way is meant to bean exhaustive list.

TABLE 3 Exemplary Pharmaceutical Agents for Conjugation CYTOTOXIC AGENTSAuristatins Auristatin E Monomethyl auristatin D (MMAD) Monomethylauristatin E (MMAE) Desmethyl auristatin E (DMAE) Auristatin FMonomethyl auristatin F (MMAF) Desmethyl auristatin F (DMAF) Auristatinderivatives, e.g., amides thereof Auristatin tyramine Auristatinquinoline Dolastatins Dolastatin derivatives Dolastatin 16 DmJDolastatin 16 Dpv Maytansinoids, e.g. DM-1; DM-4 Maytansinoidderivatives Duocarmycin Duocarmycin derivatives Alpha-amanitinAnthracyclines Doxorubicin Daunorubicin Bryostatins CamptothecinCamptothecin derivatives 7-substituted Camptothecin 10,11-Difluoromethylenedioxycamptothecin Combretastatins DebromoaplysiatoxinKahalalide-F Discodermolide Ecteinascidins ANTIVIRALS Acyclovir Vira ASymmetrel ANTIFUNGALS Nystatin ADDITIONAL ANTI-NEOPLASTICS AdriamycinCerubidine Bleomycin Alkeran Velban Oncovin Fluorouracil MethotrexateThiotepa Bisantrene Novantrone Thioguanine Procarabizine CytarabineANTI-BACTERIALS Aminoglycosides Streptomycin Neomycin Kanamycin AmikacinGentamicin Tobramycin Streptomycin B Spectinomycin AmpicillinSulfanilamide Polymyxin Chloramphenicol Turbostatin PhenstatinsHydroxyphenstatin Spongistatin 5 Spongistatin 7 Halistatin 1 Halistatin2 Halistatin 3 Modified Bryostatins Halocomstatins Pyrrolobenzimidazoles(PBI) Cibrostatin6 Doxaliform Anthracyclins analogues Cemadotin analogue(CemCH2-SH) Pseudomonas toxin A (PE38) variant Pseudomonas toxin A(ZZ-PE38) variant ZJ-101 OSW-1 4-Nitrobenzyloxycarbonyl Derivatives ofO6-Benzylguanine Topoisomerase inhibitors Hemiasterlin CephalotaxineHomoharringtonine Pyrrolobenzodiazepine dimers (PBDs) Functionalizedpyrrolobenzodiazepenes Calicheamicins Podophyllotoxins Taxanes Vincaalkaloids CONJUGATABLE DETECTION REAGENTS Fluorescein and derivativesthereof Fluorescein isothiocyanate (FITC) RADIOPHARMACEUTICALS ¹²⁵I ¹³¹I⁸⁹Zr ¹¹¹In ¹²³I ¹³¹I ⁹⁹mTc ²⁰¹Tl ¹³³Xe ¹¹C ⁶²Cu ¹⁸F ⁶⁸Ga ¹³N ¹⁵O ³⁸K⁸²Rb ⁹⁹mTc (Technetium) HEAVY METALS Barium Gold PlatinumANTI-MYCOPLASMALS Tylosine Spectinomycin

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies of thedisclosure. (See, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. In some embodiments,the binding is, however, covalent binding. Covalent binding can beachieved either by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present disclosure, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

In some embodiments, in addition to the compositions and methodsprovided herein, the conjugated activatable antibody can also bemodified for site-specific conjugation through modified amino acidsequences inserted or otherwise included in the activatable antibodysequence. These modified amino acid sequences are designed to allow forcontrolled placement and/or dosage of the conjugated agent within aconjugated activatable antibody. For example, the activatable antibodycan be engineered to include cysteine substitutions at positions onlight and heavy chains that provide reactive thiol groups and do notnegatively impact protein folding and assembly, nor alter antigenbinding. In some embodiments, the activatable antibody can be engineeredto include or otherwise introduce one or more non-natural amino acidresidues within the activatable antibody to provide suitable sites forconjugation. In some embodiments, the activatable antibody can beengineered to include or otherwise introduce enzymatically activatablepeptide sequences within the activatable antibody sequence.

Suitable linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. In someembodiments, suitable linkers include: (i) EDC(1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii)SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC. Additional linkers include, but arenot limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

In some embodiments, the linkers are cleavable. In some embodiments, thelinkers are non-cleavable. In some embodiments, two or more linkers arepresent. The two or more linkers are all the same, i.e., cleavable ornon-cleavable, or the two or more linkers are different, i.e., at leastone cleavable and at least one non-cleavable.

The present disclosure utilizes several methods for attaching agents toABs: (a) attachment to the carbohydrate moieties of the AB, or (b)attachment to sulfhydryl groups of the AB, or (c) attachment to aminogroups of the AB, or (d) attachment to carboxylate groups of the AB.According to the disclosure, ABs may be covalently attached to an agentthrough an intermediate linker having at least two reactive groups, oneto react with AB and one to react with the agent. The linker, which mayinclude any compatible organic compound, can be chosen such that thereaction with AB (or agent) does not adversely affect AB reactivity andselectivity. Furthermore, the attachment of linker to agent might notdestroy the activity of the agent. Suitable linkers for reaction withoxidized antibodies or oxidized antibody fragments include thosecontaining an amine selected from the group consisting of primary amine,secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine,semicarbazide and thiosemicarbazide groups. Such reactive functionalgroups may exist as part of the structure of the linker, or may beintroduced by suitable chemical modification of linkers not containingsuch groups.

According to the present disclosure, suitable linkers for attachment toreduced ABs include those having certain reactive groups capable ofreaction with a sulfhydryl group of a reduced antibody or fragment. Suchreactive groups include, but are not limited to: reactive haloalkylgroups (including, for example, haloacetyl groups), p-mercuribenzoategroups and groups capable of Michael-type addition reactions (including,for example, maleimides and groups of the type described by Mitra andLawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

According to the present disclosure, suitable linkers for attachment toneither oxidized nor reduced Abs include those having certain functionalgroups capable of reaction with the primary amino groups present inunmodified lysine residues in the Ab. Such reactive groups include, butare not limited to, NHS carboxylic or carbonic esters, sulfo-NHScarboxylic or carbonic esters, 4-nitrophenyl carboxylic or carbonicesters, pentafluorophenyl carboxylic or carbonic esters, acylimidazoles, isocyanates, and isothiocyanates.

According to the present disclosure, suitable linkers for attachment toneither oxidized nor reduced Abs include those having certain functionalgroups capable of reaction with the carboxylic acid groups present inaspartate or glutamate residues in the Ab, which have been activatedwith suitable reagents. Suitable activating reagents include EDC, withor without added NHS or sulfo-NHS, and other dehydrating agents utilizedfor carboxamide formation. In these instances, the functional groupspresent in the suitable linkers would include primary and secondaryamines, hydrazines, hydroxylamines, and hydrazides.

The agent may be attached to the linker before or after the linker isattached to the AB. In certain applications it may be desirable to firstproduce an AB-linker intermediate in which the linker is free of anassociated agent. Depending upon the particular application, a specificagent may then be covalently attached to the linker. In someembodiments, the AB is first attached to the MM, CM and associatedlinkers and then attached to the linker for conjugation purposes.

Branched Linkers:

In specific embodiments, branched linkers that have multiple sites forattachment of agents are utilized. For multiple site linkers, a singlecovalent attachment to an AB would result in an AB-linker intermediatecapable of binding an agent at a number of sites. The sites may bealdehyde or sulfhydryl groups or any chemical site to which agents canbe attached.

In some embodiments, higher specific activity (or higher ratio of agentsto AB) can be achieved by attachment of a single site linker at aplurality of sites on the AB. This plurality of sites may be introducedinto the AB by either of two methods. First, one may generate multiplealdehyde groups and/or sulfhydryl groups in the same AB. Second, one mayattach to an aldehyde or sulfhydryl of the AB a “branched linker” havingmultiple functional sites for subsequent attachment to linkers. Thefunctional sites of the branched linker or multiple site linker may bealdehyde or sulfhydryl groups, or may be any chemical site to whichlinkers may be attached. Still higher specific activities may beobtained by combining these two approaches, that is, attaching multiplesite linkers at several sites on the AB.

Cleavable Linkers:

Peptide linkers that are susceptible to cleavage by enzymes of thecomplement system, such as but not limited to urokinase, tissueplasminogen activator, trypsin, plasmin, or another enzyme havingproteolytic activity may be used in one embodiment of the presentdisclosure. According to one method of the present disclosure, an agentis attached via a linker susceptible to cleavage by complement. Theantibody is selected from a class that can activate complement. Theantibody-agent conjugate, thus, activates the complement cascade andreleases the agent at the target site. According to another method ofthe present disclosure, an agent is attached via a linker susceptible tocleavage by enzymes having a proteolytic activity such as a urokinase, atissue plasminogen activator, plasmin, or trypsin. These cleavablelinkers are useful in conjugated activatable antibodies that include anextracellular toxin, e.g., by way of non-limiting example, any of theextracellular toxins shown in Table 3.

Non-limiting examples of cleavable linker sequences are provided inTable 4.

TABLE 4 Exemplary Linker Sequences for Conjugation Types of CleavableSequences Amino Acid Sequence Plasmin cleavable sequences Pro-urokinasePRFKIIGG (SEQ ID NO: 127) PRFRIIGG (SEQ ID NO: 128) TGFβ SSRHRRALD (SEQID NO: 129) Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 130) StaphylokinaseSSSFDKGKYKKGDDA (SEQ ID NO: 131) SSSFDKGKYKRGDDA (SEQ ID NO: 132) FactorXa cleavable sequences IEGR (SEQ ID NO: 133) IDGR (SEQ ID NO: 134)GGSIDGR (SEQ ID NO: 135) MMP cleavable sequences Gelatinase A PLGLWA(SEQ ID NO: 136) Collagenase cleavable sequences Calf skin collagen(α1(I) chain) GPQGIAGQ (SEQ ID NO: 137) Calf skin collagen (α2(I) chain)GPQGLLGA (SEQ ID NO: 138) Bovine cartilage collagen (α1(II) chain) GIAGQ(SEQ ID NO: 139) Human liver collagen (α1(III) chain) GPLGIAGI (SEQ IDNO: 140) Human α₂M GPEGLRVG (SEQ ID NO: 141) Human PZP YGAGLGVV (SEQ IDNO: 142) AGLGVVER (SEQ ID NO: 143) AGLGISST (SEQ ID NO: 144) Rat α₁MEPQALAMS (SEQ ID NO: 145) QALAMSAI (SEQ ID NO: 146) Rat α₂M AAYHLVSQ(SEQ ID NO: 147) MDAFLESS (SEQ ID NO: 148) Rat α₁I₃(2J) ESLPVVAV (SEQ IDNO: 149) Rat α₁I₃(27J) SAPAVESE (SEQ ID NO: 150) Human fibroblastcollagenase DVAQFVLT (SEQ ID NO: 151) (autolytic cleavages) VAQFVLTE(SEQ ID NO: 152) AQFVLTEG (SEQ ID NO: 153) PVQPIGPQ (SEQ ID NO: 154)

In addition, agents may be attached via disulfide bonds (for example,the disulfide bonds on a cysteine molecule) to the AB. Since many tumorsnaturally release high levels of glutathione (a reducing agent) this canreduce the disulfide bonds with subsequent release of the agent at thesite of delivery. In certain specific embodiments, the reducing agentthat would modify a CM would also modify the linker of the conjugatedactivatable antibody.

Spacers and Cleavable Elements:

In some embodiments, it may be necessary to construct the linker in sucha way as to optimize the spacing between the agent and the AB of theactivatable antibody. This may be accomplished by use of a linker of thegeneral structure:

W—(CH₂)n-Q

whereinW is either —NH—CH₂— or —CH₂—;Q is an amino acid, peptide; andn is an integer from 0 to 20.

In some embodiments, the linker may comprise a spacer element and acleavable element. The spacer element serves to position the cleavableelement away from the core of the AB such that the cleavable element ismore accessible to the enzyme responsible for cleavage. Certain of thebranched linkers described above may serve as spacer elements.

Throughout this discussion, it should be understood that the attachmentof linker to agent (or of spacer element to cleavable element, orcleavable element to agent) need not be particular mode of attachment orreaction. Any reaction providing a product of suitable stability andbiological compatibility is acceptable.

Serum Complement and Selection of Linkers:

According to one method of the present disclosure, when release of anagent is desired, an AB that is an antibody of a class that can activatecomplement is used. The resulting conjugate retains both the ability tobind antigen and activate the complement cascade. Thus, according tothis embodiment of the present disclosure, an agent is joined to one endof the cleavable linker or cleavable element and the other end of thelinker group is attached to a specific site on the AB. For example, ifthe agent has an hydroxy group or an amino group, it may be attached tothe carboxy terminus of a peptide, amino acid or other suitably chosenlinker via an ester or amide bond, respectively. For example, suchagents may be attached to the linker peptide via a carbodimide reaction.If the agent contains functional groups that would interfere withattachment to the linker, these interfering functional groups can beblocked before attachment and deblocked once the product conjugate orintermediate is made. The opposite or amino terminus of the linker isthen used either directly or after further modification for binding toan AB that is capable of activating complement.

Linkers (or spacer elements of linkers) may be of any desired length,one end of which can be covalently attached to specific sites on the ABof the activatable antibody. The other end of the linker or spacerelement may be attached to an amino acid or peptide linker.

Thus when these conjugates bind to antigen in the presence of complementthe amide or ester bond that attaches the agent to the linker will becleaved, resulting in release of the agent in its active form. Theseconjugates, when administered to a subject, will accomplish delivery andrelease of the agent at the target site, and are particularly effectivefor the in vivo delivery of pharmaceutical agents, antibiotics,antimetabolites, antiproliferative agents and the like as presented inbut not limited to those in Table 3.

Linkers for Release without Complement Activation:

In yet another application of targeted delivery, release of the agentwithout complement activation is desired since activation of thecomplement cascade will ultimately lyse the target cell. Hence, thisapproach is useful when delivery and release of the agent should beaccomplished without killing the target cell. Such is the goal whendelivery of cell mediators such as hormones, enzymes, corticosteroids,neurotransmitters, genes or enzymes to target cells is desired. Theseconjugates may be prepared by attaching the agent to an AB that is notcapable of activating complement via a linker that is mildly susceptibleto cleavage by serum proteases. When this conjugate is administered toan individual, antigen-antibody complexes will form quickly whereascleavage of the agent will occur slowly, thus resulting in release ofthe compound at the target site.

Biochemical Cross Linkers:

In some embodiments, the activatable antibody may be conjugated to oneor more therapeutic agents using certain biochemical cross-linkers.Cross-linking reagents form molecular bridges that tie togetherfunctional groups of two different molecules. To link two differentproteins in a step-wise manner, hetero-bifunctional cross-linkers can beused that eliminate unwanted homopolymer formation.

Peptidyl linkers cleavable by lysosomal proteases are also useful, forexample, Val-Cit, Val-Ala or other dipeptides. In addition, acid-labilelinkers cleavable in the low-pH environment of the lysosome may be used,for example: bis-sialyl ether. Other suitable linkers includecathepsin-labile substrates, particularly those that show optimalfunction at an acidic pH.

Exemplary hetero-bifunctional cross-linkers are referenced in Table 5.

TABLE 5 Exemplary Hetero-Bifunctional Cross Linkers HETERO-BIFUNCTIONALCROSS-LINKERS Spacer Arm Length after Reactive Advantages andcross-linking Linker Toward Applications (Angstroms) SMPT Primary aminesGreater stability 11.2 Å Sulfhydryls SPDP Primary amines Thiolation  6.8Å Sulfhydryls Cleavable cross-linking LC-SPDP Primary amines Extendedspacer arm 15.6 Å Sulfhydryls Sulfo-LC- Primary amines Extender spacerarm 15.6 Å SPDP Sulfhydryls Water-soluble SMCC Primary amines Stablemaleimide reactive 11.6 Å group Sulfhydryls Enzyme-antibody conjugationHapten-carrier proteinconjugation Sulfo-SMCC Primary amines Stablemaleimide reactive 11.6 Å group Sulfhydryls Water-solubleEnzyme-antibody conjugation MBS Primary amines Enzyme-antibody  9.9 ÅSulfhydryls conjugation Hapten-carrier protein conjugation Sulfo-MBSPrimary amines Water-soluble  9.9 Å Sulfhydryls SIAB Primary aminesEnzyme-antibody 10.6 Å Sulfhydryls conjugation Sulfo-SIAB Primary aminesWater-soluble 10.6 Å Sulfhydryls SMPB Primary amines Extended spacer arm14.5 Å Sulfhydryls Enzyme-antibody conjugation Sulfo-SMPB Primary aminesExtended spacer arm 14.5 Å Sulfhydryls Water-soluble EDE/Sulfo- Primaryamines Hapten-Carrier conjugation 0 NHS Carboxyl groups ABHCarbohydrates Reacts with sugar groups 11.9 Å Nonselective

Non-Cleavable Linkers or Direct Attachment:

In some embodiments of the disclosure, the conjugate may be designed sothat the agent is delivered to the target but not released. This may beaccomplished by attaching an agent to an AB either directly or via anon-cleavable linker.

These non-cleavable linkers may include amino acids, peptides, D-aminoacids or other organic compounds that may be modified to includefunctional groups that can subsequently be utilized in attachment to ABsby the methods described herein. A-general formula for such an organiclinker could be

W—(CH₂)n-Q

whereinW is either —NH—CH₂— or —CH₂—;Q is an amino acid, peptide; andn is an integer from 0 to 20.

Non-Cleavable Conjugates:

In some embodiments, a compound may be attached to ABs that do notactivate complement. When using ABs that are incapable of complementactivation, this attachment may be accomplished using linkers that aresusceptible to cleavage by activated complement or using linkers thatare not susceptible to cleavage by activated complement.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present disclosure canbe conjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. The term “a”entity or “an” entity refers to one or more of that entity. For example,a compound refers to one or more compounds. As such, the terms “a”,“an”, “one or more” and “at least one” can be used interchangeably.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “immunospecifically bind” is meant that theantibody reacts with one or more antigenic determinants of the desiredantigen and does not react with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab,and F(ab′)₂ fragments, scFvs, and an Fab expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. For example, antibodies may be raisedagainst N-terminal or C-terminal peptides of a polypeptide. An antibodyis said to specifically bind an antigen when the dissociation constantis ≦1 μM; in some embodiments, ≦100 nM and in some embodiments, ≦10 nM.

As used herein, the terms “specific binding,” “immunological binding,”and “immunological binding properties” refer to the non-covalentinteractions of the type which occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. The strength,or affinity of immunological binding interactions can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. Immunological bindingproperties of selected polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(K_(on)) and the “off rate constant” (K_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation. (See Nature 361:186-87 (1993)). The ratio ofK_(off)/K_(on) enables the cancellation of all parameters not related toaffinity, and is equal to the dissociation constant K_(d). (See,generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). Anantibody of the present disclosure is said to specifically bind to thetarget, when the equilibrium binding constant (K_(d)) is ≦1 μM, in someembodiments 100 nM, in some embodiments ≦10 nM, and in some embodiments≦100 pM to about 1 pM, as measured by assays such as radioligand bindingassays or similar assays known to those skilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the disclosure include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules shown herein, andnucleic acid molecules encoding the light chain immunoglobulin moleculesshown herein.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of murine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the disclosure comprise the heavychain immunoglobulin molecules shown herein, and the light chainimmunoglobulin molecules shown herein, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andthat has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences that are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein meansnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term oligonucleotide referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. In some embodiments, oligonucleotides are10 to 60 bases in length and in some embodiments, 12, 13, 14, 15, 16,17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usuallysingle stranded, e.g., for probes, although oligonucleotides may bedouble stranded, e.g., for use in the construction of a gene mutant.Oligonucleotides of the disclosure are either sense or antisenseoligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotide linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such asα-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids may also be suitable components forpolypeptides of the present disclosure. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and thatare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and that are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, in some embodiments, at least 90 percent sequenceidentity, in some embodiments, at least 95 percent sequence identity,and in some embodiments, at least 99 percent sequence identity.

In some embodiments, residue positions that are not identical differ byconservative amino acid substitutions.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present disclosure, providing that the variations inthe amino acid sequence maintain at least 75%, in some embodiments, atleast 80%, 90%, 95%, and in some embodiments, 99%. In particular,conservative amino acid replacements are contemplated. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare generally divided into families: (1) acidic amino acids areaspartate, glutamate; (2) basic amino acids are lysine, arginine,histidine; (3) non-polar amino acids are alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan, and (4)uncharged polar amino acids are glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. The hydrophilic amino acidsinclude arginine, asparagine, aspartate, glutamine, glutamate,histidine, lysine, serine, and threonine. The hydrophobic amino acidsinclude alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine and valine. Other familiesof amino acids include (i) serine and threonine, which are thealiphatic-hydroxy family; (ii) asparagine and glutamine, which are theamide containing family; (iii) alanine, valine, leucine and isoleucine,which are the aliphatic family; and (iv) phenylalanine, tryptophan, andtyrosine, which are the aromatic family. For example, it is reasonableto expect that an isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid will not have a major effect on the binding or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional peptide can readily be determined by assaying thespecific activity of the polypeptide derivative. Assays are described indetail herein. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.Suitable amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. In some embodiments,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the disclosure.

Suitable amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (5) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions (forexample, conservative amino acid substitutions) may be made in thenaturally-occurring sequence (for example, in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion and/or oneor more internal deletion(s), but where the remaining amino acidsequence is identical to the corresponding positions in thenaturally-occurring sequence deduced, for example, from a full lengthcDNA sequence. Fragments typically are at least 5, 6, 8 or 10 aminoacids long, in some embodiments, at least 14 amino acids long, in someembodiments, at least 20 amino acids long, usually at least 50 aminoacids long, and in some embodiments, at least 70 amino acids long. Theterm “analog” as used herein refers to polypeptides that are comprisedof a segment of at least 25 amino acids that has substantial identity toa portion of a deduced amino acid sequence and that has specific bindingto the target, under suitable binding conditions. Typically, polypeptideanalogs comprise a conservative amino acid substitution (or addition ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, in some embodiments, atleast 50 amino acids long or longer, and can often be as long as afull-length naturally-occurring polypeptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and in someembodiments, a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, in some embodiments, more than about 85%, 90%, 95%, and99%. In some embodiments, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Activatable antibodies of the disclosure specifically bind a giventarget, e.g., a human target protein. Also included in the disclosureare activatable antibodies that bind to the same epitope as theactivatable antibodies described herein.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody(e.g., a murine monoclonal or humanized antibody) has the samespecificity as a monoclonal antibody used in the methods describedherein by ascertaining whether the former prevents the latter frombinding to the target. If the monoclonal antibody being tested competeswith the monoclonal antibody of the disclosure, as shown by a decreasein binding by the monoclonal antibody of the disclosure, then the twomonoclonal antibodies bind to the same, or a closely related, epitope. Amethod for determining whether a monoclonal antibody has the specificityof a monoclonal antibody of the disclosure is to pre-incubate themonoclonal antibody of the disclosure with the target and then add themonoclonal antibody being tested to determine if the monoclonal antibodybeing tested is inhibited in its ability to bind the target. If themonoclonal antibody being tested is inhibited then, in all likelihood,it has the same, or functionally equivalent, epitopic specificity as themonoclonal antibody of the disclosure.

Multispecific Activatable Antibodies

The disclosure also provides multispecific activatable antibodies. Themultispecific activatable antibodies provided herein are multispecificantibodies that recognize two or more different antigens or epitopes andthat include at least one masking moiety (MM) linked to at least oneantigen- or epitope-binding domain of the multispecific antibody suchthat coupling of the MM reduces the ability of the antigen- orepitope-binding domain to bind its target. In some embodiments, the MMis coupled to the antigen- or epitope-binding domain of themultispecific antibody via a cleavable moiety (CM) that functions as asubstrate for at least one MMP protease. The activatable multispecificantibodies provided herein are stable in circulation, activated atintended sites of therapy and/or diagnosis but not in normal, i.e.,healthy tissue, and, when activated, exhibit binding to a target that isat least comparable to the corresponding, unmodified multispecificantibody.

In some embodiments, the multispecific activatable antibodies aredesigned to engage immune effector cells, also referred to herein asimmune-effector cell engaging multispecific activatable antibodies. Insome embodiments, the multispecific activatable antibodies are designedto engage leukocytes, also referred to herein as leukocyte engagingmultispecific activatable antibodies. In some embodiments, themultispecific activatable antibodies are designed to engage T cells,also referred to herein as T-cell engaging multispecific activatableantibodies. In some embodiments, the multispecific activatableantibodies engage a surface antigen on a leukocyte, such as on a T cell,on a natural killer (NK) cell, on a myeloid mononuclear cell, on amacrophage, and/or on another immune effector cell. In some embodiments,the immune effector cell is a leukocyte. In some embodiments, the immuneeffector cell is a T cell. In some embodiments, the immune effector cellis a NK cell. In some embodiments, the immune effector cell is amononuclear cell, such as a myeloid mononuclear cell. In someembodiments, the multispecific activatable antibodies are designed tobind or otherwise interact with more than one target and/or more thanone epitope, also referred to herein as multi-antigen targetingactivatable antibodies. As used herein, the terms “target” and “antigen”are used interchangeably.

In some embodiments, immune effector cell engaging multispecificactivatable antibodies of the disclosure include a targeting antibody orantigen-binding fragment thereof and an immune effector cell engagingantibody or antigen-binding portion thereof, where at least one of thetargeting antibody or antigen-binding fragment thereof and/or the immuneeffector cell engaging antibody or antigen-binding portion thereof ismasked. In some embodiments, the immune effector cell engaging antibodyor antigen binding fragment thereof includes a first antibody orantigen-binding fragment thereof (AB1) that binds a first, immuneeffector cell engaging target, where the AB1 is attached to a maskingmoiety (MM1) such that coupling of the MM1 reduces the ability of theAB1 to bind the first target. In some embodiments, the targetingantibody or antigen-binding fragment thereof includes a second antibodyor fragment thereof that includes a second antibody or antigen-bindingfragment thereof (AB2) that binds a second target, where the AB2 isattached to a masking moiety (MM2) such that coupling of the MM2 reducesthe ability of the AB2 to bind the second target. In some embodiments,the immune effector cell engaging antibody or antigen binding fragmentthereof includes a first antibody or antigen-binding fragment thereof(AB1) that binds a first, immune effector cell engaging target, wherethe AB1 is attached to a masking moiety (MM1) such that coupling of theMM1 reduces the ability of the AB1 to bind the first target, and thetargeting antibody or antigen-binding fragment thereof includes a secondantibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target. In someembodiments, the non-immune effector cell engaging antibody is a cancertargeting antibody. In some embodiments the non-immune cell effectorantibody is an IgG. In some embodiments the immune effector cellengaging antibody is a scFv. In some embodiments the targeting antibody(e.g., non-immune cell effector antibody) is an IgG and the immuneeffector cell engaging antibody is a scFv. In some embodiments, theimmune effector cell is a leukocyte. In some embodiments, the immuneeffector cell is a T cell. In some embodiments, the immune effector cellis a NK cell. In some embodiments, the immune effector cell is a myeloidmononuclear cell.

In some embodiments, T-cell engaging multispecific activatableantibodies of the disclosure include a targeting antibody orantigen-binding fragment thereof and a T-cell engaging antibody orantigen-binding portion thereof, where at least one of the targetingantibody or antigen-binding fragment thereof and/or the T-cell engagingantibody or antigen-binding portion thereof is masked. In someembodiments, the T-cell engaging antibody or antigen binding fragmentthereof includes a first antibody or antigen-binding fragment thereof(AB1) that binds a first, T-cell engaging target, where the AB1 isattached to a masking moiety (MM1) such that coupling of the MM1 reducesthe ability of the AB1 to bind the first target. In some embodiments,the targeting antibody or antigen-binding fragment thereof includes asecond antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target. In someembodiments, the T-cell engaging antibody or antigen binding fragmentthereof includes a first antibody or antigen-binding fragment thereof(AB1) that binds a first, T-cell engaging target, where the AB1 isattached to a masking moiety (MM1) such that coupling of the MM1 reducesthe ability of the AB1 to bind the first target, and the targetingantibody or antigen-binding fragment thereof includes a second antibodyor fragment thereof that includes a second antibody or antigen-bindingfragment thereof (AB2) that binds a second target, where the AB2 isattached to a masking moiety (MM2) such that coupling of the MM2 reducesthe ability of the AB2 to bind the second target.

In some embodiments, the T-cell engaging multispecific activatableantibodies include a cancer targeting antibody or antigen-bindingfragment thereof and a T-cell engaging antibody or antigen-bindingportion thereof, where at least one of the cancer targeting antibody orantigen-binding fragment thereof and/or the T-cell engaging antibody orantigen-binding portion thereof is masked. In some embodiments, theT-cell engaging antibody or antigen binding fragment thereof includes afirst antibody or antigen-binding fragment thereof (AB1) that binds afirst, T-cell engaging target, where the AB1 is attached to a maskingmoiety (MM1) such that coupling of the MM1 reduces the ability of theAB1 to bind the first target. In some embodiments, the cancer targetingantibody or antigen-binding fragment thereof includes a second antibodyor fragment thereof that includes a second antibody or antigen-bindingfragment thereof (AB2) that binds a second, cancer-related target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second, cancer-relatedtarget. In some embodiments, the T-cell engaging antibody or antigenbinding fragment thereof includes a first antibody or antigen-bindingfragment thereof (AB1) that binds a first, T-cell engaging target, wherethe AB1 is attached to a masking moiety (MM1) such that coupling of theMM1 reduces the ability of the AB1 to bind the first target, and thecancer targeting antibody or antigen-binding fragment thereof includes asecond antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second,cancer-related target, where the AB2 is attached to a masking moiety(MM2) such that coupling of the MM2 reduces the ability of the AB2 tobind the second, cancer-related target.

In some embodiments, the T-cell engaging multispecific activatableantibodies include a cancer targeting IgG antibody or antigen-bindingfragment thereof and a T-cell engaging scFv, where at least one of thecancer targeting IgG antibody or antigen-binding fragment thereof and/orthe T-cell engaging antibody or antigen-binding portion thereof ismasked. In some embodiments, the T-cell engaging antibody or antigenbinding fragment thereof includes a first antibody or antigen-bindingfragment thereof (AB1) that binds a first, T-cell engaging target, wherethe AB1 is attached to a masking moiety (MM1) such that coupling of theMM1 reduces the ability of the AB1 to bind the first target. In someembodiments, the cancer targeting IgG antibody or antigen-bindingfragment thereof includes a second antibody or fragment thereof thatincludes a second antibody or antigen-binding fragment thereof (AB2)that binds a second, cancer-related target, where the AB2 is attached toa masking moiety (MM2) such that coupling of the MM2 reduces the abilityof the AB2 to bind the second, cancer-related target. In someembodiments, the T-cell engaging antibody or antigen binding fragmentthereof includes a first antibody or antigen-binding fragment thereof(AB1) that binds a first, T-cell engaging target, where the AB1 isattached to a masking moiety (MM1) such that coupling of the MM1 reducesthe ability of the AB1 to bind the first target, and the cancertargeting IgG antibody or antigen-binding fragment thereof includes asecond antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second,cancer-related target, where the AB2 is attached to a masking moiety(MM2) such that coupling of the MM2 reduces the ability of the AB2 tobind the second, cancer-related target.

In some embodiments of an immune effector cell engaging multispecificactivatable antibody, one antigen is typically an antigen present on thesurface of a tumor cell or other cell type associated with disease, suchas, but not limited to, any target listed in Table 1, such as, but notlimited to, EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71(transferrin receptor), and another antigen is typically a stimulatoryor inhibitory receptor present on the surface of a T-cell, naturalkiller (NK) cell, myeloid mononuclear cell, macrophage, and/or otherimmune effector cell, such as, but not limited to, B7-H4, BTLA, CD3,CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR,HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. In someembodiments, the antigen is a stimulatory receptor present on thesurface of a T cell or NK cell; examples of such stimulatory receptorsinclude, but are not limited to, CD3, CD27, CD28, CD137 (also referredto as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In some embodiments,the antigen is an inhibitory receptor present on the surface of aT-cell; examples of such inhibitory receptors include, but are notlimited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressedKIRs. The antibody domain conferring specificity to the T-cell surfaceantigen may also be substituted by a ligand or ligand domain that bindsto a T-cell receptor, a NK-cell receptor, a macrophage receptor, and/orother immune effector cell receptor, such as, but not limited to, B7-1,B7-2, B7H3, PD-L1, PD-L2, or TNFSF9.

One embodiment of the disclosure is a multispecific activatable antibodythat is activatable in a cancer microenvironment and that includes anantibody, for example a IgG or scFv, directed to a tumor target and anagonist antibody, for example an IgG or scFv, directed to aco-stimulatory receptor expressed on the surface of an activated T cellor NK cell, wherein at least one of the cancer target antibody and/oragonist antibody is masked. Examples of co-stimulatory receptorsinclude, but are not limited to, CD27, CD137, GITR, HVEM, NKG2D, andOX40. In this embodiment, the multispecific activatable antibody, onceactivated by tumor-associated proteases, would effectively crosslink andactivate the T cell or NK cell expressed co-stimulatory receptors in atumor-dependent manner to enhance the activity of T cells that areresponding to any tumor antigen via their endogenous T cell antigen orNK-activating receptors. The activation-dependent nature of these T cellor NK cell costimulatory receptors would focus the activity of theactivated multispecific activatable antibody to tumor-specific T cells,without activating all T cells independent of their antigen specificity.In one embodiment, at least the co-stimulatory receptor antibody of themultispecific activatable antibody is masked to prevent activation ofauto-reactive T cells that may be present in tissues that also expressthe antigen recognized by the tumor target-directed antibody in themultispecific activatable antibody, but whose activity is restricted bylack of co-receptor engagement.

One embodiment of the disclosure is a multispecific activatable antibodythat is activatable in a disease characterized by T celloverstimulation, such as, but not limited to, an autoimmune disease orinflammatory disease microenvironment. Such a multispecific activatableantibody includes an antibody, for example a IgG or scFv, directed to atarget comprising a surface antigen expressed in a tissue targeted by aT cell in autoimmune or inflammatory disease and an antibody, forexample a IgG or scFv, directed to an inhibitory receptor expressed onthe surface of a T cell or NK cell, wherein at least one of the diseasetissue target antibody and/or T cell inhibitory receptor antibody ismasked. Examples of inhibitory receptors include, but are not limitedto, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIRs.Examples of a tissue antigen targeted by T cells in autoimmune diseaseinclude, but are not limited to, a surface antigen expressed on myelinor nerve cells in multiple sclerosis or a surface antigen expressed onpancreatic islet cells in Type 1 diabetes. In this embodiment, themultispecific activatable antibody when localized in the tissue underautoimmune attack or inflammation is activated and co-engages the T cellor NK cell inhibitory receptor to suppress the activity of autoreactiveT cells responding to any disease tissue-targeted antigens via theirendogenous TCR or activating receptors. In one embodiment, at least oneor multiple antibodies are masked to prevent suppression of T cellresponses in non-disease tissues where the target antigen may also beexpressed.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CD3 epsilon (CD3ε, also referred to herein asCD3e and CD3) scFv and a targeting antibody or antigen-binding fragmentthereof, where at least one of the anti-CD3ε scFv and/or the targetingantibody or antigen-binding portion thereof is masked. In someembodiments, the CD3ε scFv includes a first antibody or antigen-bindingfragment thereof (AB1) that binds CD3ε, where the AB1 is attached to amasking moiety (MM1) such that coupling of the MM1 reduces the abilityof the AB1 to bind CD3ε. In some embodiments, the targeting antibody orantigen-binding fragment thereof includes a second antibody or fragmentthereof that includes a second antibody or antigen-binding fragmentthereof (AB2) that binds a second target, where the AB2 is attached to amasking moiety (MM2) such that coupling of the MM2 reduces the abilityof the AB2 to bind the second target. In some embodiments, the CD3εscFvincludes a first antibody or antigen-binding fragment thereof (AB1) thatbinds CD3ε, where the AB1 is attached to a masking moiety (MM1) suchthat coupling of the MM1 reduces the ability of the AB1 to bind CD3ε,and the targeting antibody or antigen-binding fragment thereof includesa second antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CD3ε scFv and a cancer targeting antibody orantigen-binding fragment thereof, where at least one of the anti-CD3εscFv and/or the cancer targeting antibody or antigen-binding portionthereof is masked. In some embodiments, the CD3ε scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε. In someembodiments, the cancer targeting antibody or antigen-binding fragmentthereof includes a second antibody or fragment thereof that includes asecond antibody or antigen-binding fragment thereof (AB2) that binds asecond, cancer-related target, where the AB2 is attached to a maskingmoiety (MM2) such that coupling of the MM2 reduces the ability of theAB2 to bind the second, cancer-related target. In some embodiments, theCD3ε scFv includes a first antibody or antigen-binding fragment thereof(AB1) that binds CD3ε, where the AB1 is attached to a masking moiety(MM1) such that coupling of the MM1 reduces the ability of the AB1 tobind CD3ε, and the cancer targeting antibody or antigen-binding fragmentthereof includes a second antibody or fragment thereof that includes asecond antibody or antigen-binding fragment thereof (AB2) that binds asecond, cancer-related target, where the AB2 is attached to a maskingmoiety (MM2) such that coupling of the MM2 reduces the ability of theAB2 to bind the second, cancer-related target.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CD3ε scFv and a cancer targeting IgG antibodyor antigen-binding fragment thereof, where at least one of the anti-CD3εscFv and/or the cancer targeting IgG antibody or antigen-binding portionthereof is masked. In some embodiments, the CD3ε scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε. In someembodiments, the cancer targeting IgG antibody or antigen-bindingfragment thereof includes a second antibody or fragment thereof thatincludes a second antibody or antigen-binding fragment thereof (AB2)that binds a second, cancer-related target, where the AB2 is attached toa masking moiety (MM2) such that coupling of the MM2 reduces the abilityof the AB2 to bind the second, cancer-related target. In someembodiments, the CD3ε scFv includes a first antibody or antigen-bindingfragment thereof (AB1) that binds CD3ε, where the AB1 is attached to amasking moiety (MM1) such that coupling of the MM1 reduces the abilityof the AB1 to bind CD3ε, and the cancer targeting antibody IgG orantigen-binding fragment thereof includes a second antibody or fragmentthereof that includes a second antibody or antigen-binding fragmentthereof (AB2) that binds a second, cancer-related target, where the AB2is attached to a masking moiety (MM2) such that coupling of the MM2reduces the ability of the AB2 to bind the second, cancer-relatedtarget.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CD3 epsilon (CD3ε) scFv that is derived fromOKT3, where at least one of the targeting antibody or antigen-bindingfragment thereof and/or the OKT3 scFv or OKT3-derived scFv is masked. Insome embodiments, the OKT3 scFv or OKT3-derived scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε. In someembodiments, the targeting antibody or antigen-binding fragment thereofincludes a second antibody or fragment thereof that includes a secondantibody or antigen-binding fragment thereof (AB2) that binds a secondtarget, where the AB2 is attached to a masking moiety (MM2) such thatcoupling of the MM2 reduces the ability of the AB2 to bind the secondtarget. In some embodiments, the OKT3 scFv or OKT3-derived scFv includesa first antibody or antigen-binding fragment thereof (AB1) that bindsCD3ε, where the AB1 is attached to a masking moiety (MM1) such thatcoupling of the MM1 reduces the ability of the AB1 to bind CD3ε, and thetargeting antibody or antigen-binding fragment thereof includes a secondantibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an OKT3 scFv or OKT3-derived scFv and a cancertargeting antibody or antigen-binding fragment thereof, where at leastone of the OKT3 scFv or OKT3-derived scFv and/or the cancer targetingantibody or antigen-binding portion thereof is masked. In someembodiments, the OKT3 scFv or OKT3-derived scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε. In someembodiments, the cancer targeting antibody or antigen-binding fragmentthereof includes a second antibody or fragment thereof that includes asecond antibody or antigen-binding fragment thereof (AB2) that binds asecond, cancer-related target, where the AB2 is attached to a maskingmoiety (MM2) such that coupling of the MM2 reduces the ability of theAB2 to bind the second, cancer-related target. In some embodiments, theOKT3 scFv or OKT3-derived scFv includes a first antibody orantigen-binding fragment thereof (AB1) that binds CD3ε, where the AB1 isattached to a masking moiety (MM1) such that coupling of the MM1 reducesthe ability of the AB1 to bind CD3ε, and the cancer targeting antibodyor antigen-binding fragment thereof includes a second antibody orfragment thereof that includes a second antibody or antigen-bindingfragment thereof (AB2) that binds a second, cancer-related target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second, cancer-relatedtarget.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an OKT3 scFv or OKT3-derived scFv and a cancertargeting IgG antibody or antigen-binding fragment thereof, where atleast one of the OKT3 scFv or OKT3-derived scFv and/or the cancertargeting IgG antibody or antigen-binding portion thereof is masked. Insome embodiments, the OKT3 scFv or OKT3-derived scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε. In someembodiments, the cancer targeting IgG antibody or antigen-bindingfragment thereof includes a second antibody or fragment thereof thatincludes a second antibody or antigen-binding fragment thereof (AB2)that binds a second, cancer-related target, where the AB2 is attached toa masking moiety (MM2) such that coupling of the MM2 reduces the abilityof the AB2 to bind the second, cancer-related target. In someembodiments, the OKT3 scFv or OKT3-derived scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε, and the cancertargeting antibody IgG or antigen-binding fragment thereof includes asecond antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second,cancer-related target, where the AB2 is attached to a masking moiety(MM2) such that coupling of the MM2 reduces the ability of the AB2 tobind the second, cancer-related target.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CTLA-4 scFv, where at least one of thetargeting antibody or antigen-binding fragment thereof and/or theanti-CTLA-4 scFv is masked. In some embodiments, the anti-CTLA-4 scFvincludes a first antibody or antigen-binding fragment thereof (AB1) thatbinds CTLA-4, where the AB1 is attached to a masking moiety (MM1) suchthat coupling of the MM1 reduces the ability of the AB1 to bind CTLA-4.In some embodiments, the targeting antibody or antigen-binding fragmentthereof includes a second antibody or fragment thereof that includes asecond antibody or antigen-binding fragment thereof (AB2) that binds asecond target, where the AB2 is attached to a masking moiety (MM2) suchthat coupling of the MM2 reduces the ability of the AB2 to bind thesecond target. In some embodiments, the anti-CTLA-4 scFv includes afirst antibody or antigen-binding fragment thereof (AB1) that bindsCTLA-4, where the AB1 is attached to a masking moiety (MM1) such thatcoupling of the MM1 reduces the ability of the AB1 to bind CTLA-4, andthe targeting antibody or antigen-binding fragment thereof includes asecond antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CTLA-4 scFv and a targeting IgG antibody orantigen-binding fragment thereof, where at least one of the anti-CTLA-4scFv and/or the targeting IgG antibody or antigen-binding portionthereof is masked. In some embodiments, the anti-CTLA-4 scFv includes afirst antibody or antigen-binding fragment thereof (AB1) that bindsCTLA-4, where the AB1 is attached to a masking moiety (MM1) such thatcoupling of the MM1 reduces the ability of the AB1 to bind CTLA-4. Insome embodiments, the targeting IgG antibody or antigen-binding fragmentthereof includes a second antibody or fragment thereof that includes asecond antibody or antigen-binding fragment thereof (AB2) that binds asecond target, where the AB2 is attached to a masking moiety (MM2) suchthat coupling of the MM2 reduces the ability of the AB2 to bind thesecond target. In some embodiments, the anti-CTLA-4 scFv includes afirst antibody or antigen-binding fragment thereof (AB1) that bindsCTLA-4, where the AB1 is attached to a masking moiety (MM1) such thatcoupling of the MM1 reduces the ability of the AB1 to bind CTLA-4, andthe targeting antibody IgG or antigen-binding fragment thereof includesa second antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target.

In some embodiments, the multi-antigen targeting antibodies and/ormulti-antigen targeting activatable antibodies include at least a firstantibody or antigen-binding fragment thereof that binds a first targetand/or first epitope and a second antibody or antigen-binding fragmentthereof that binds a second target and/or a second epitope. In someembodiments, the multi-antigen targeting antibodies and/or multi-antigentargeting activatable antibodies bind two or more different targets. Insome embodiments, the multi-antigen targeting antibodies and/ormulti-antigen targeting activatable antibodies bind two or moredifferent epitopes on the same target. In some embodiments, themulti-antigen targeting antibodies and/or multi-antigen targetingactivatable antibodies bind a combination of two or more differenttargets and two or more different epitopes on the same target.

In some embodiments, a multispecific activatable antibody comprising anIgG has the IgG variable domains masked. In some embodiments, amultispecific activatable antibody comprising a scFv has the scFvdomains masked. In some embodiments, a multispecific activatableantibody has both IgG variable domains and scFv domains, where at leastone of the IgG variable domains is coupled to a masking moiety. In someembodiments, a multispecific activatable antibody has both IgG variabledomains and scFv domains, where at least one of the scFv domains iscoupled to a masking moiety. In some embodiments, a multispecificactivatable antibody has both IgG variable domains and scFv domains,where at least one of the IgG variable domains is coupled to a maskingmoiety and at least one of the scFv domains is coupled to a maskingmoiety. In some embodiments, a multispecific activatable antibody hasboth IgG variable domains and scFv domains, where each of the IgGvariable domains and the scFv domains is coupled to its own maskingmoiety. In some embodiments, one antibody domain of a multispecificactivatable antibody has specificity for a target antigen and anotherantibody domain has specificity for a T-cell surface antigen. In someembodiments, one antibody domain of a multispecific activatable antibodyhas specificity for a target antigen and another antibody domain hasspecificity for another target antigen. In some embodiments, oneantibody domain of a multispecific activatable antibody has specificityfor an epitope of a target antigen and another antibody domain hasspecificity for another epitope of the target antigen.

In a multispecific activatable antibody, a scFv can be fused to thecarboxyl terminus of the heavy chain of an IgG activatable antibody, tothe carboxyl terminus of the light chain of an IgG activatable antibody,or to the carboxyl termini of both the heavy and light chains of an IgGactivatable antibody. In a multispecific activatable antibody, a scFvcan be fused to the amino terminus of the heavy chain of an IgGactivatable antibody, to the amino terminus of the light chain of an IgGactivatable antibody, or to the amino termini of both the heavy andlight chains of an IgG activatable antibody. In a multispecificactivatable antibody, a scFv can be fused to any combination of one ormore carboxyl termini and one or more amino termini of an IgGactivatable antibody. In some embodiments, a masking moiety (MM) linkedto a cleavable moiety (CM) is attached to and masks an antigen bindingdomain of the IgG. In some embodiments, a masking moiety (MM) linked toa cleavable moiety (CM) is attached to and masks an antigen bindingdomain of at least one scFv. In some embodiments, a masking moiety (MM)linked to a cleavable moiety (CM) is attached to and masks an antigenbinding domain of an IgG and a masking moiety (MM) linked to a cleavablemoiety (CM) is attached to and masks an antigen binding domain of atleast one scFv.

The disclosure provides examples of multispecific activatable antibodystructures which include, but are not limited to, the following:(VL-CL)₂:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*-L2-CM-L1-MM)₂;(VL-CL)₂:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2-CM-L1-MM)₂;(MM-L1-CM-L2-VL-CL)₂: (VH-CH1-CH2-CH3-L4-VH*-L3-VL*)₂;(MM-L1-CM-L2-VL-CL)₂:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*)₂;(VL-CL)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂;(VL-CL)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂;(MM-L1-CM-L2-VL-CL)₂: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂;(MM-L1-CM-L2-VL-CL)₂:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂: (VH-CH1-CH2-CH3)₂;(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂: (VH-CH1-CH2-CH3)₂;(MM-L1-CM-L2-VL*-L3-VH*-L4-VL-CL)₂:(VH-CH1-CH2-CH3)₂;(MM-L1-CM-L2-VH*-L3-VL*-L4-VL-CL)₂: (VH-CH1-CH2-CH3)₂;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂: (VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; or(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂: (VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂,wherein: VL and VH represent the light and heavy variable domains of thefirst specificity, contained in the IgG; VL* and VH* represent thevariable domains of the second specificity, contained in the scFv; L1 isa linker peptide connecting the masking moiety (MM) and the cleavablemoiety (CM); L2 is a linker peptide connecting the cleavable moiety(CM), and the antibody; L3 is a linker peptide connecting the variabledomains of the scFv; L4 is a linker peptide connecting the antibody ofthe first specificity to the antibody of the second specificity; CL isthe light-chain constant domain; and CH1, CH2, CH3 are the heavy chainconstant domains. The first and second specificities may be toward anyantigen or epitope.

In some embodiments of a T-cell engaging multispecific activatableantibody, one antigen is typically an antigen present on the surface ofa tumor cell or other cell type associated with disease, such as, butnot limited to, any target listed in Table 1, such as, but not limitedto, EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrinreceptor), and another antigen is typically a stimulatory (also referredto herein as activating) or inhibitory receptor present on the surfaceof a T-cell, natural killer (NK) cell, myeloid mononuclear cell,macrophage, and/or other immune effector cell, such as, but not limitedto, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56,CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3,NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain conferringspecificity to the T-cell surface antigen may also be substituted by aligand or ligand domain that binds to a T-cell receptor, a NK-cellreceptor, a macrophage receptor, and/or other immune effector cellreceptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2,or TNFSF9. In some embodiments of a multi-antigen targeting activatableantibody, one antigen is selected from the group of targets listed inTable 1, and another antigen is selected from the group of targetslisted in Table 1.

In some embodiments, the targeting antibody is an anti-EGFR antibody. Insome embodiments, the targeting antibody is C225v5, which is specificfor binding to EGFR. In some embodiments, the targeting antibody isC225, which is specific for binding to EGFR. In some embodiments, thetargeting antibody is C225v4, which is specific for binding to EGFR. Insome embodiments, the targeting antibody is C225v6, which is specificfor binding to EGFR. In some embodiments, the targeting antibody is ananti-Jagged antibody. In some embodiments, the targeting antibody is4D11, which is specific for binding to human and mouse Jagged 1 andJagged 2. In some embodiments, the targeting antibody is 4D11v2, whichis specific for binding to human and mouse Jagged 1 and Jagged 2.

In some embodiments, the targeting antibody can be in the form anactivatable antibody. In some embodiments, the scFv(s) can be in theform of a Pro-scFv (see, e.g., WO 2009/025846, WO 2010/081173).

In some embodiments, the scFv is specific for binding CD3ε, and is or isderived from an antibody or fragment thereof that binds CD3ε, e.g.,CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. In some embodiments, thescFv is specific for binding CTLA-4 (also referred to herein as CTLA andCTLA4).

In some embodiments, the anti-CTLA-4 scFv includes the amino acidsequence:

(SEQ ID NO: 510) GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRGTLVTVSSAS

In some embodiments, the anti-CTLA-4 scFv includes the amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence of SEQ ID NO: 510.

In some embodiments, the anti-CD3ε scFv includes the amino acidsequence:

(SEQ ID NO: 511) GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSN PFTFGSGTKLEINR

In some embodiments, the anti-CD3ε scFv includes the amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 511.

In some embodiments, the scFv is specific for binding one or moreT-cells, one or more NK-cells and/or one or more macrophages. In someembodiments, the scFv is specific for binding a target selected from thegroup consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28,CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1,TIGIT, TIM3, or VISTA.

In some embodiments, the multispecific activatable antibody alsoincludes an agent conjugated to the AB. In some embodiments, the agentis a therapeutic agent. In some embodiments, the agent is anantineoplastic agent. In some embodiments, the agent is a toxin orfragment thereof. In some embodiments, the agent is conjugated to themultispecific activatable antibody via a linker. In some embodiments,the agent is conjugated to the AB via a cleavable linker. In someembodiments, the agent is conjugated to the AB via a linker thatincludes at least one MMP-cleavable substrate sequence. In someembodiments, the linker is a non-cleavable linker. In some embodiments,the agent is a microtubule inhibitor. In some embodiments, the agent isa nucleic acid damaging agent, such as a DNA alkylator or DNAintercalator, or other DNA damaging agent. In some embodiments, thelinker is a cleavable linker. In some embodiments, the agent is an agentselected from the group listed in Table 4. In some embodiments, theagent is a dolastatin. In some embodiments, the agent is an auristatinor derivative thereof. In some embodiments, the agent is auristatin E ora derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid ormaytansinoid derivative. In some embodiments, the agent is DM1 or DM4.In some embodiments, the agent is a duocarmycin or derivative thereof.In some embodiments, the agent is a calicheamicin or derivative thereof.In some embodiments, the agent is a pyrrolobenzodiazepine.

In some embodiments, the multispecific activatable antibody alsoincludes a detectable moiety. In some embodiments, the detectable moietyis a diagnostic agent.

In some embodiments, the multispecific activatable antibody naturallycontains one or more disulfide bonds. In some embodiments, themultispecific activatable antibody can be engineered to include one ormore disulfide bonds.

The disclosure also provides an isolated nucleic acid molecule encodinga multispecific activatable antibody described herein, as well asvectors that include these isolated nucleic acid sequences. Thedisclosure provides methods of producing a multispecific activatableantibody by culturing a cell under conditions that lead to expression ofthe activatable antibody, wherein the cell comprises such a nucleic acidmolecule. In some embodiments, the cell comprises such a vector.

The disclosure also provides a method of manufacturing multispecificactivatable antibodies of the disclosure by (a) culturing a cellcomprising a nucleic acid construct that encodes the multispecificactivatable antibody under conditions that lead to expression of themultispecific activatable, and (b) recovering the multispecificactivatable antibody.

The disclosure also provides multispecific activatable antibodies and/ormultispecific activatable antibody compositions that include at least afirst antibody or antigen-binding fragment thereof (AB1) thatspecifically binds a first target or first epitope and a second antibodyor antigen-biding fragment thereof (AB2) that binds a second target or asecond epitope, where at least AB1 is coupled or otherwise attached to amasking moiety (MM1), such that coupling of the MM1 reduces the abilityof AB1 to bind its target. In some embodiments, the MM1 is coupled toAB1 via a first cleavable moiety (CM1) sequence that includes asubstrate for a protease, for example, a protease that is co-localizedwith the target of AB1 at a treatment site or a diagnostic site in asubject. The multispecific activatable antibodies provided herein arestable in circulation, activated at intended sites of therapy and/ordiagnosis but not in normal, i.e., healthy tissue, and, when activated,exhibit binding to the target of AB1 that is at least comparable to thecorresponding, unmodified multispecific antibody.

In some embodiments, the multispecific activatable antibody comprises alinking peptide between the MM1 and the CM1.

In some embodiments, the multispecific activatable antibody comprises alinking peptide between the CM1 and the AB1.

In some embodiments, the activatable antibody comprises a first linkingpeptide (LP1) and a second linking peptide (LP2), and at least a portionof the multispecific activatable antibody has the structural arrangementfrom N-terminus to C-terminus as follows in the uncleaved state:MM1-LP1-CM1-LP2-AB1 or AB1-LP2-CM1-LP1-MM1. In some embodiments, the twolinking peptides need not be identical to each other.

In some embodiments, at least one of LP1 or LP2 includes an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 1) and (GGGS)_(n) (SEQ ID NO: 2), where n is aninteger of at least one. In some embodiments, at least one of LP1 or LP2includes an amino acid sequence selected from the group consisting ofGGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG(SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8).

In some embodiments, the multispecific activatable antibody includes atleast a first antibody or antigen-binding fragment thereof (AB1) thatspecifically binds a first target or first epitope and a second antibodyor antigen-binding fragment thereof (AB2) that specifically binds asecond target or second epitope. In some embodiments, each of the AB inthe multispecific activatable antibody is independently selected fromthe group consisting of a monoclonal antibody, domain antibody, singlechain, Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb, a dAb, a singledomain heavy chain antibody, and a single domain light chain antibody.In some embodiments, each of the AB in the multispecific activatableantibody is a rodent (e.g., mouse or rat), chimeric, humanized or fullyhuman monoclonal antibody.

In some embodiments, each of the AB in the multispecific activatableantibody has an equilibrium dissociation constant of about 100 nM orless for binding to its corresponding target or epitope.

In some embodiments, MM1 has an equilibrium dissociation constant forbinding to its corresponding AB that is greater than the equilibriumdissociation constant of the AB to its corresponding target or epitope.

In some embodiments, MM1 has an equilibrium dissociation constant forbinding to its corresponding AB that is no more than the equilibriumdissociation constant of the AB to its corresponding target or epitope.

In some embodiments, MM1 does not interfere or compete with itscorresponding AB for binding to the corresponding target or epitope whenthe multispecific activatable antibody is in a cleaved state.

In some embodiments, MM1 is a polypeptide of about 2 to 40 amino acidsin length. In some embodiments, each of the MM in the multispecificactivatable antibody is a polypeptide of no more than 40 amino acids inlength.

In some embodiments, MM1 has a polypeptide sequence that is differentfrom that of target of the corresponding AB.

In some embodiments, MM1 has a polypeptide sequence that is no more than50% identical to any natural binding partner of the corresponding AB. Insome embodiments, MM1 has a polypeptide sequence that is no more than25% identical to any natural binding partner of the corresponding AB. Insome embodiments, MM1 has a polypeptide sequence that is no more than10% identical to any natural binding partner of the corresponding AB.

In some embodiments, the coupling of MM1 reduces the ability of thecorresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM1 towardsits corresponding target or epitope is at least 20 times greater thanthe K_(d) of the AB when not coupled to the MM1 towards itscorresponding target or epitope.

In some embodiments, the coupling of MM1 reduces the ability of thecorresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM1 towardsits corresponding target or epitope is at least 40 times greater thanthe K_(d) of the AB when not coupled to the MM1 towards itscorresponding target or epitope.

In some embodiments, the coupling of MM1 reduces the ability of thecorresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM1 towardsits corresponding target or epitope is at least 100 times greater thanthe K_(d) of the AB when not coupled to the MM1 towards itscorresponding target or epitope.

In some embodiments, the coupling of MM1 reduces the ability of thecorresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM1 towardsits corresponding target or epitope is at least 1000 times greater thanthe K_(d) of the AB when not coupled to the MM1 towards itscorresponding target or epitope.

In some embodiments, the coupling of MM1 reduces the ability of thecorresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM1 towardsits corresponding target or epitope is at least 10,000 times greaterthan the K_(d) of the AB when not coupled to the MM1 towards itscorresponding target or epitope.

In some embodiments, MM1 is an amino acid sequence selected from a MMdisclosed herein.

In some embodiments, the multispecific activatable antibody includes atleast a second masking moiety (MM2) that inhibits the binding of the AB2to its target when the multispecific activatable antibody is in anuncleaved state, and a second cleavable moiety (CM2) coupled to the AB2,wherein the CM2 is a polypeptide that functions as a substrate for asecond protease. In some embodiments, CM2 is a polypeptide of no morethan 15 amino acids long. In some embodiments, the second protease isco-localized with the second target or epitope in a tissue, and whereinthe second protease cleaves the CM2 in the multispecific activatableantibody when the multispecific activatable antibody is exposed to thesecond protease. In some embodiments, the first protease and the secondprotease are co-localized with the first target or epitope and thesecond target or epitope in a tissue. In some embodiments, the firstprotease and the second protease are the same protease. In someembodiments, CM1 and CM2 are different substrates for the same protease.In some embodiments, the protease is selected from the group consistingof those shown in Table 7. In some embodiments, the first protease andthe second protease are different proteases. In some embodiments, thefirst protease and the second protease are different proteases selectedfrom the group consisting of those shown in Table 7.

In some embodiments, each of the MM in the multispecific activatableantibody, e.g., MM1 and at least MM2, has an equilibrium dissociationconstant for binding to its corresponding AB that is greater than theequilibrium dissociation constant of the AB to its corresponding targetor epitope.

In some embodiments, each of the MM in the multispecific activatableantibody has an equilibrium dissociation constant for binding to itscorresponding AB that is no more than the equilibrium dissociationconstant of the AB to its corresponding target or epitope.

In some embodiments, each of the MM in the multispecific activatableantibody does not interfere or compete with its corresponding AB forbinding to the corresponding target or epitope when the multispecificactivatable antibody is in a cleaved state.

In some embodiments, each of the MM in the multispecific activatableantibody is a polypeptide of about 2 to 40 amino acids in length. Insome embodiments, each of the MM in the multispecific activatableantibody is a polypeptide of no more than 40 amino acids in length.

In some embodiments, each of the MM in the multispecific activatableantibody has a polypeptide sequence that is different from that oftarget of the corresponding AB.

In some embodiments, each of the MM in the multispecific activatableantibody has a polypeptide sequence that is no more than 50% identicalto any natural binding partner of the corresponding AB. In someembodiments, each of the MM in the multispecific activatable antibodyhas a polypeptide sequence that is no more than 25% identical to anynatural binding partner of the corresponding AB. In some embodiments,each of the MM in the multispecific activatable antibody has apolypeptide sequence that is no more than 10% identical to any naturalbinding partner of the corresponding AB.

In some embodiments, the coupling of each of the MM reduces the abilityof the corresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsits corresponding target or epitope is at least 20 times greater thanthe K_(d) of the AB when not coupled to the MM towards its correspondingtarget or epitope.

In some embodiments, the coupling of each of the MM reduces the abilityof the corresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsits corresponding target or epitope is at least 40 times greater thanthe K_(d) of the AB when not coupled to the MM towards its correspondingtarget or epitope.

In some embodiments, the coupling of each of the MM reduces the abilityof the corresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsits corresponding target or epitope is at least 100 times greater thanthe K_(d) of the AB when not coupled to the MM towards its correspondingtarget or epitope.

In some embodiments, the coupling of each of the MM reduces the abilityof the corresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsits corresponding target or epitope is at least 1000 times greater thanthe K_(d) of the AB when not coupled to the MM towards its correspondingtarget or epitope.

In some embodiments, the coupling of each of the MM reduces the abilityof the corresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsits corresponding target or epitope is at least 10,000 times greaterthan the K_(d) of the AB when not coupled to the MM towards itscorresponding target or epitope.

In some embodiments, each of the MM is an amino acid sequence selectedfrom a MM disclosed herein.

In some embodiments, at least one of CM1 and/or CM2 is cleaved by atleast one MMP protease. In some embodiments, at least one of CM1 and/orCM2 includes an amino acid sequence selected from the group consistingof ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID NO: 15); AQNLLGMV (SEQ IDNO: 16); STFPFGMF (SEQ ID NO: 17); PVGYTSSL (SEQ ID NO: 18); DWLYWPGI(SEQ ID NO: 19); MIAPVAYR (SEQ ID NO: 20); RPSPMWAY (SEQ ID NO: 21);WATPRPMR (SEQ ID NO: 22); FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQ ID NO:24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG (SEQID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29); LAAPLGLL(SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO: 32);PAGLWLDP (SEQ ID NO: 33); and ISSGLSS (SEQ ID NO: 159).

In some embodiments, at least one of CM1 and/or CM2 includes an aminoacid sequence selected from the group consisting of SEQ ID NOs: 364-370,379-393, 402-409, 420-424, 434, 435, 450-452, 457, 470-472, 474, and483.

In some embodiments, at least one of CM1 and/or CM2 includes an aminoacid sequence selected from the group consisting of SEQ ID NOs: 328,336-339, and 348-351.

In some embodiments, the protease that cleaves the first cleavablemoiety (CM1) sequence is co-localized with the target of the AB1 in themultispecific activatable antibody in a tissue, and the protease cleavesthe CM1 in the multispecific activatable antibody when the multispecificactivatable antibody is exposed to the protease.

In some embodiments, the multispecific activatable antibody includesmore than one cleavable moiety sequence, and the protease that cleavesat least one cleavable moiety sequence is co-localized with the targetof at least one of the AB regions in the multispecific activatableantibody in a tissue, and the protease cleaves the CM in themultispecific activatable antibody when the multispecific activatableantibody is exposed to the protease.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least twofold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least threefold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least fourfold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least fivefold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least tenfold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least 20-fold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM is positioned in the multispecificactivatable antibody such that in the uncleaved state, binding of themultispecific activatable antibody to a target of one of the AB regionsis reduced to occur with an equilibrium dissociation constant that is atleast 40-fold greater than the equilibrium dissociation constant of anunmodified AB binding to its target, and whereas in the cleaved state,the AB binds its target.

In some embodiments, each CM is positioned in the multispecificactivatable antibody such that in the uncleaved state, binding of themultispecific activatable antibody to a target of one of the AB regionsis reduced to occur with an equilibrium dissociation constant that is atleast 50-fold greater than the equilibrium dissociation constant of anunmodified AB binding to its target, and whereas in the cleaved state,the AB binds its target.

In some embodiments, each CM is positioned in the multispecificactivatable antibody such that in the uncleaved state, binding of themultispecific activatable antibody to a target of one of the AB regionsis reduced to occur with an equilibrium dissociation constant that is atleast 100-fold greater than the equilibrium dissociation constant of anunmodified AB binding to its target, and whereas in the cleaved state,the AB binds its target.

In some embodiments, each CM is positioned in the multispecificactivatable antibody such that in the uncleaved state, binding of themultispecific activatable antibody to a target of one of the AB regionsis reduced to occur with an equilibrium dissociation constant that is atleast 200-fold greater than the equilibrium dissociation constant of anunmodified AB binding to its target, and whereas in the cleaved state,the AB binds its target.

In some embodiments, each CM in the multispecific activatable antibodyis a polypeptide of up to 15 amino acids in length.

In some embodiments, at least one CM in the multispecific activatableantibody includes an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14-33 and 159 and the other CM includes theamino acid sequence LSGRSDNH (SEQ ID NO: 26). In some embodiments, atleast one CM includes the amino acid sequence LSGRSDNH (SEQ ID NO: 26).In some embodiments, at least one cleavable moiety is selected for usewith a specific protease, for example a protease that is known to beco-localized with at least one target of the multispecific activatableantibody. For example, suitable cleavable moieties for use in themultispecific activatable antibodies of the disclosure are cleaved by atleast a protease such as urokinase, legumain, and/or matriptase (alsoreferred to herein as MT-SP1 or MTSP1). In some embodiments, a suitablecleavable moiety includes at least one of the following sequences:TGRGPSWV (SEQ ID NO: 27); SARGPSRW (SEQ ID NO: 28); TARGPSFK (SEQ ID NO:29); LSGRSDNH (SEQ ID NO: 26); GGWHTGRN (SEQ ID NO: 30); HTGRSGAL (SEQID NO: 31); PLTGRSGG (SEQ ID NO: 32); AARGPAIH (SEQ ID NO: 33); RGPAFNPM(SEQ ID NO: 34); SSRGPAYL (SEQ ID NO: 35); RGPATPIM (SEQ ID NO: 36);RGPA (SEQ ID NO: 37); GGQPSGMWGW (SEQ ID NO: 38); FPRPLGITGL (SEQ ID NO:39); VHMPLGFLGP (SEQ ID NO: 40); SPLTGRSG (SEQ ID NO: 41); SAGFSLPA (SEQID NO: 42); LAPLGLQRR (SEQ ID NO: 43); SGGPLGVR (SEQ ID NO: 44); and/orPLGL (SEQ ID NO: 45).

In some embodiments, one CM is a substrate for at least one MMP proteaseand the other CM in the multispecific activatable antibody is asubstrate for a protease selected from the group consisting of thoseshown in Table 7. In some embodiments, the protease is selected from thegroup consisting of uPA, legumain, matriptase, ADAM17, BMP-1, TMPRSS3,TMPRSS4, neutrophil elastase, MMP-7, MMP-9, MMP-12, MMP-13, and MMP-14.In some embodiments, the protease is a cathepsin, such as, but notlimited to, cathepsin S. In some embodiments, each CM in themultispecific activatable antibody is a substrate for a proteaseselected from the group consisting of uPA (urokinase plasminogenactivator), legumain and matriptase. In some embodiments, the proteasecomprises uPA. In some embodiments, the protease comprises legumain. Insome embodiments, the protease comprises matriptase. In someembodiments, the protease comprises a matrix metalloproteinase (MMP).

In some embodiments, at least one CM in the multispecific activatableantibody is a substrate for at least two proteases. In some embodiments,each protease is selected from the group consisting of those shown inTable 7. In some embodiments, at least one CM in the multispecificactivatable antibody is a substrate for at least two proteases, whereinone of the proteases is selected from the group consisting of uPA,legumain and matriptase and the other protease is selected from thegroup consisting of those shown in Table 7. In some embodiments, atleast one CM in the multispecific activatable antibody is a substratefor at least two proteases selected from the group consisting of uPA,legumain and matriptase.

In some embodiments, the multispecific activatable antibody includes atleast a first CM (CM1) and a second CM (CM2). In some embodiments, CM1and CM2 are part of a single cleavable linker that joins an MM to an AB.In some embodiments, CM1 is part of a cleavable linker that joins MM1 toAB1, and CM2 is part of a separate cleavable linker that joins an MM2 toAB2. In some embodiments, a multispecific activatable antibody comprisesmore than two CMs. In some embodiments, such a multispecific activatableantibody comprises more than two CMs and more than two MMs. In someembodiments, CM1 and CM2 are each polypeptides of no more than 15 aminoacids long. In some embodiments, at least one of the first CM and thesecond CM is a polypeptide that functions as a substrate for a proteaseselected from the group consisting of those listed in Table 7. In someembodiments, at least one of the first CM and the second CM is apolypeptide that functions as a substrate for a protease selected fromthe group consisting of uPA, legumain, and matriptase. In someembodiments, the first CM is cleaved by a first cleaving agent selectedfrom the group consisting of uPA, legumain, and matriptase in a targettissue and the second CM is cleaved by a second cleaving agent in atarget tissue. In some embodiments, the other protease is selected fromthe group consisting of those shown in Table 7. In some embodiments, thefirst cleaving agent and the second cleaving agent are the same proteaseselected from the group consisting of those listed in Table 7, and thefirst CM and the second CM are different substrates for the enzyme. Insome embodiments, the first cleaving agent and the second cleaving agentare the same protease selected from the group consisting of uPA,legumain, and matriptase, and the first CM and the second CM aredifferent substrates for the enzyme. In some embodiments, the firstcleaving agent and the second cleaving agent are the same proteaseselected from the group listed in Table 7, and the first CM and thesecond CM are the same substrate. In some embodiments, the firstcleaving agent and the second cleaving agent are different proteases. Insome embodiments, the first cleaving agent and the second cleaving agentare different proteases selected from the group consisting of thoseshown in Table 7. In some embodiments, the first cleaving agent and thesecond cleaving agent are co-localized in the target tissue. In someembodiments, the first CM and the second CM are cleaved by at least onecleaving agent in the target tissue.

In some embodiments, the multispecific activatable antibody is exposedto and cleaved by a protease such that, in the activated or cleavedstate, the activated multispecific activatable antibody includes a lightchain amino acid sequence that includes at least a portion of LP2 and/orCM sequence after the protease has cleaved the CM.

The disclosure also provides compositions and methods that include amultispecific activatable antibody that includes at least a firstantibody or antibody fragment (AB1) that specifically binds a target anda second antibody or antibody fragment (AB2), where at least the firstAB in the multispecific activatable antibody is coupled to a maskingmoiety (MM1) that decreases the ability of AB1 to bind its target. Insome embodiments, each AB is coupled to a MM that decreases the abilityof its corresponding AB to each target. For example, in bispecificactivatable antibody embodiments, AB1 is coupled to a first maskingmoiety (MM1) that decreases the ability of AB1 to bind its target, andAB2 is coupled to a second masking moiety (MM2) that decreases theability of AB2 to bind its target. In some embodiments, themultispecific activatable antibody comprises more than two AB regions;in such embodiments, AB1 is coupled to a first masking moiety (MM1) thatdecreases the ability of AB1 to bind its target, AB2 is coupled to asecond masking moiety (MM2) that decreases the ability of AB2 to bindits target, AB3 is coupled to a third masking moiety (MM3) thatdecreases the ability of AB3 to bind its target, and so on for each ABin the multispecific activatable antibody.

In some embodiments, the multispecific activatable antibody furtherincludes at least one cleavable moiety (CM) that is a substrate for aprotease, where the CM links a MM to an AB. For example, in someembodiments, the multispecific activatable antibody includes at least afirst antibody or antibody fragment (AB1) that specifically binds atarget and a second antibody or antibody fragment (AB2), where at leastthe first AB in the multispecific activatable antibody is coupled via afirst cleavable moiety (CM1) to a masking moiety (MM1) that decreasesthe ability of AB1 to bind its target. In some bispecific activatableantibody embodiments, AB1 is coupled via CM1 to MM1, and AB2 is coupledvia a second cleavable moiety (CM2) to a second masking moiety (MM2)that decreases the ability of AB2 to bind its target. In someembodiments, the multispecific activatable antibody comprises more thantwo AB regions; in some of these embodiments, AB1 is coupled via CM1 toMM1, AB2 is coupled via CM2 to MM2, and AB3 is coupled via a thirdcleavable moiety (CM3) to a third masking moiety (MM3) that decreasesthe ability of AB3 to bind its target, and so on for each AB in themultispecific activatable antibody.

Activatable Antibodies Having Non-Binding Steric Moieties or BindingPartners for Non-Binding Steric Moieties

The disclosure also provides activatable antibodies that includenon-binding steric moieties (NB) or binding partners (BP) fornon-binding steric moieties, where the BP recruits or otherwise attractsthe NB to the activatable antibody. The activatable antibodies providedherein include, for example, an activatable antibody that includes anon-binding steric moiety (NB), a cleavable linker (CL) and antibody orantibody fragment (AB) that binds a target; an activatable antibody thatincludes a binding partner for a non-binding steric moiety (BP), a CLand an AB; and an activatable antibody that includes a BP to which an NBhas been recruited, a CL and an AB that binds the target. Activatableantibodies in which the NB is covalently linked to the CL and AB of theactivatable antibody or is associated by interaction with a BP that iscovalently linked to the CL and AB of the activatable antibody arereferred to herein as “NB-containing activatable antibodies.” Byactivatable or switchable is meant that the activatable antibodyexhibits a first level of binding to a target when the activatableantibody is in an inhibited, masked or uncleaved state (i.e., a firstconformation), and a second level of binding to the target when theactivatable antibody is in an uninhibited, unmasked and/or cleaved state(i.e., a second conformation, i.e., activated antibody), where thesecond level of target binding is greater than the first level of targetbinding. The activatable antibody compositions can exhibit increasedbioavailability and more favorable biodistribution compared toconventional antibody therapeutics.

In some embodiments, activatable antibodies provide for reduced toxicityand/or adverse side effects that could otherwise result from binding ofthe at non-treatment sites and/or non-diagnostic sites if the AB werenot masked or otherwise inhibited from binding to such a site.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to the target, wherein the NB is apolypeptide that does not bind specifically to the AB; the CL is apolypeptide that includes a substrate (S) for an enzyme; the CL ispositioned such that in an uncleaved state, the NB interferes withbinding of the AB to the target and in a cleaved state, the NB does notinterfere with binding of the AB to the target; and the NB does notinhibit cleavage of the CL by the enzyme. As used herein and throughout,the term polypeptide refers to any polypeptide that includes at leasttwo amino acid residues, including larger polypeptides, full-lengthproteins and fragments thereof, and the term polypeptide is not limitedto single-chain polypeptides and can include multi-unit, e.g.,multi-chain, polypeptides. In cases where the polypeptide is of ashorter length, for example, less than 50 amino acids total, the termspeptide and polypeptide are used interchangeably herein, and in caseswhere the polypeptide is of a longer length, e.g., 50 amino acids orgreater, the terms polypeptide and protein are used interchangeablyherein.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to the target, wherein (i) the NBincludes a polypeptide that does not bind specifically to the AB; (ii)CL is a polypeptide of up to 50 amino acids in length that includes asubstrate (S) for an enzyme; (iii) the CL is positioned such that in anuncleaved state, the NB interferes with binding of the AB to the targetand in a cleaved state, the NB does not interfere with binding of the ABto the target; and (iv) the NB does not inhibit cleavage of the CL bythe enzyme. For example, the CL has a length of up to 15 amino acids, alength of up to 20 amino acids, a length of up to 25 amino acids, alength of up to 30 amino acids, a length of up to 35 amino acids, alength of up to 40 amino acids, a length of up to 45 amino acids, alength of up to 50 amino acids, a length in the range of 10-50 aminoacids, a length in the range of 15-50 amino acids, a length in the rangeof 20-50 amino acids, a length in the range of 25-50 amino acids, alength in the range of 30-50 amino acids, a length in the range of 35-50amino acids, a length in the range of 40-50 amino acids, a length in therange of 45-50 amino acids, a length in the range of 10-40 amino acids,a length in the range of 15-40 amino acids, a length in the range of20-40 amino acids, a length in the range of 25-40 amino acids, a lengthin the range of 30-40 amino acids, a length in the range of 35-40 aminoacids, a length in the range of 10-30 amino acids, a length in the rangeof 15-30 amino acids, a length in the range of 20-30 amino acids, alength in the range of 25-30 amino acids, a length in the range of 10-20amino acids, or a length in the range of 10-15 amino acids.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to the target, wherein (i) the NBincludes a polypeptide that does not bind specifically to the AB; (ii)the CL is a polypeptide that includes a substrate (S) for an enzyme;(iii) the CL is positioned such that in an uncleaved state, the NBinterferes with binding of the AB to the target and in a cleaved state,the NB does not interfere with binding of the AB to the target; (iv) theNB does not inhibit cleavage of the CL by the enzyme; and (v) theactivatable antibody has the structural arrangement from N-terminus toC-terminus as follows in the uncleaved state: NB-CL-AB or AB-CL-NB.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to the target, wherein (i) the NBincludes a polypeptide that does not bind specifically to the AB; (ii)the CL is a polypeptide that includes a substrate (S) for an enzyme;(iii) the CL is positioned such that in an uncleaved state, the NBinterferes with binding of the AB to the target and in a cleaved state,the NB does not interfere with binding of the AB to the target, andwherein the NB in the uncleaved activatable antibody reduces the abilityof the AB to bind the target by at least 50%, for example, by at least60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%,by at least 90%, by at least 95%, by at least 96%, by at least 97%, byat least 98%, by at least 99%, by at least 100% as compared to theability of the cleaved AB to bind the target; and (iv) the NB does notinhibit cleavage of the CL by the enzyme. The reduction in the abilityof the AB to bind the target is determined, e.g., using an assay asdescribed herein or an in vitro target displacement assay such as, forexample, the assay described in PCT Publication Nos. WO 2009/025846 andWO 2010/081173.

In one embodiment, the activatable antibody includes a binding partner(BP) for a non-binding steric moiety (NB); a cleavable linker (CL); andan antibody or antibody fragment (AB) that binds specifically to thetarget, wherein the BP is a polypeptide that binds to the NB whenexposed thereto; the NB does not bind specifically to the AB; the CL isa polypeptide that includes a substrate (S) for an enzyme; the CL ispositioned such that in an uncleaved state in the presence of the NB,the NB interferes with binding of the AB to the target and in a cleavedstate, the NB does not interfere with binding of the AB to the targetand the BP does not interfere with binding of the AB to the target; andthe NB and the BP do not inhibit cleavage of the CL by the enzyme. Insome examples of this embodiment, the BP of the activatable antibody isoptionally bound to the NB. In one embodiment, the NB is recruited bythe BP of the activatable antibody in vivo.

In some examples of any of these activatable antibody embodiments, theactivatable antibody is formulated as a composition. In some of theseembodiments, the composition also includes the NB, where the NB isco-formulated with the activatable antibody that includes the BP, theCL, and the AB. In some examples of this embodiment, the BP is selectedfrom the group consisting of an albumin binding peptide, a fibrinogenbinding peptide, a fibronectin binding peptide, a hemoglobin bindingpeptide, a transferrin binding peptide, an immunoglobulin domain bindingpeptide, and other serum protein binding peptides.

In some examples of any of these activatable antibody embodiments, theNB is a soluble, globular protein. In some examples of any of theseactivatable antibody embodiments, the NB is a protein that circulates inthe bloodstream. In some examples of any of these activatable antibodyembodiments, the NB is selected from the group consisting of albumin,fibrinogen, fibronectin, hemoglobin, transferrin, an immunoglobulindomain, and other serum proteins.

In some examples of any of these activatable antibody embodiments, theCL is a polypeptide that includes a substrate (S) for a protease. Insome examples of any of these activatable antibody embodiments, theprotease is co-localized with the in a tissue, and the protease cleavesthe CL in the activatable antibody when the activatable antibody isexposed to the protease. In some examples of any of these activatableantibody embodiments, the CL is a polypeptide of up to 50 amino acids inlength. In some examples of any of these activatable antibodyembodiments, the CL is a polypeptide that includes a substrate (S)having a length of up to 15 amino acids, e.g., 3 amino acids long, 4amino acids long, 5 amino acids long, 6 amino acids long, 7 amino acidslong, 8 amino acids long, 9 amino acids long, 10 amino acids long, 11amino acids long, 12 amino acids long, 13 amino acids long, 14 aminoacids long, or 15 amino acids long.

In some examples of any of these activatable antibody embodiments, theactivatable antibody has the structural arrangement from N-terminus toC-terminus as follows in the uncleaved state: NB-CL-AB, AB-CL-NB,BP-CL-AB or AB-CL-BP. In embodiments where the activatable antibodyincludes a BP and the activatable antibody is in the presence of thecorresponding NB, the activatable antibody has a structural arrangementfrom N-terminus to C-terminus as follows in the uncleaved state:NB:BP-CM-AB or AB-CM-BP:NB, where “:” represents an interaction, e.g.,binding, between the NB and BP.

In some examples of any of these activatable antibody embodiments, theactivatable antibody includes an antibody or antigen-binding fragmentthereof that specifically binds a given target and is a monoclonalantibody, domain antibody, single chain, Fab fragment, a F(ab′)₂fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody,or a single domain light chain antibody. In some embodiments, such anantibody or immunologically active fragment thereof that binds thetarget a mouse, other rodent, chimeric, humanized or fully humanmonoclonal antibody.

In some examples of any of these activatable antibody embodiments, theactivatable antibody includes a combination of a variable heavy chainregion comprising an amino acid sequence presented herein and a variablelight chain region comprising an amino acid sequence presented herein.In some embodiments, the activatable antibody includes a combination ofa variable heavy chain region comprising an amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to an amino acid sequence presented herein, and a variablelight chain region comprising an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to anamino acid sequence presented herein.

In some examples of any of these activatable antibody embodiments, theactivatable antibody also includes an agent conjugated to the AB. Insome embodiments, the agent is a therapeutic agent. In some embodiments,the agent is an antineoplastic agent. In some embodiments, the agent isa toxin or fragment thereof. In some embodiments, the agent isconjugated to the AB via a linker. In some embodiments, the linker is acleavable linker. In some embodiments, the agent is conjugated to the ABvia a noncleavable linker. In some embodiments, the agent is an agentselected from the group listed in Table 3. In some embodiments, theagent is a microtubule inhibitor. In some embodiments, the agent is anucleic acid damaging agent, such as a DNA alkylator or DNAintercalator, or other DNA damaging agent. In some embodiments, theagent is a dolastatin. In some embodiments, the agent is an auristatinor derivative thereof. In some embodiments, the agent is auristatin E ora derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid ormaytansinoid derivative. In some embodiments, the agent is DM1 or DM4.In some embodiments, the agent is a duocarmycin or derivative thereof.In some embodiments, the agent is a calicheamicin or derivative thereof.In some embodiments, the agent is a pyrrolobenzodiazepine.

In some examples of any of these activatable antibody embodiments, theactivatable antibody also includes a detectable moiety. In someembodiments, the detectable moiety is a diagnostic agent.

In some examples of any of these activatable antibody embodiments, theactivatable antibody also includes a spacer. In some examples of any ofthese activatable antibody embodiments, the activatable antibody alsoincludes a signal peptide. In some embodiments, the signal peptide isconjugated to the activatable antibody via a spacer. In some examples ofany of these activatable antibody embodiments, the spacer is joineddirectly to the MM of the activatable antibody.

In some embodiments, the serum half-life of the activatable antibody islonger than that of the corresponding antibody; e.g., the pK of theactivatable antibody is longer than that of the corresponding antibody.In some embodiments, the serum half-life of the activatable antibody issimilar to that of the corresponding antibody. In some embodiments, theserum half-life of the activatable antibody is at least 15 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 12 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 11 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least10 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 9 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 8 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 7 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least6 days when administered to an organism. In some examples of any ofthese activatable antibody embodiments, the serum half-life of theactivatable antibody is at least 5 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 4 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least3 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 2 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 24 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 20 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least18 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 16 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 14 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 12 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least10 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 8 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 6 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 4 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least3 hours when administered to an organism.

The disclosure also provides an isolated nucleic acid molecule encodingany of these activatable antibodies, as well as vectors that includethese isolated nucleic acid sequences. The disclosure provides methodsof producing an activatable antibody by culturing a cell underconditions that lead to expression of the activatable antibody, whereinthe cell comprises such a nucleic acid sequence. In some embodiments,the cell comprises such a vector.

The dissociation constant (K_(d)) of the NB-containing activatableantibody toward the target is greater than the K_(d) of the AB towardsthe target when it is not associated with the NB or NB:BP. Thedissociation constant (K_(d)) of the NB-containing activatable antibodytoward the target is greater than the K_(d) of the parental AB towardsthe target. For example, the K_(d) of the NB-containing activatableantibody toward the target is at least 5, 10, 25, 50, 100, 250, 500,1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100,10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000,10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or100,000-10,000,000 times greater than the K_(d) of the AB when it is notassociated with the NB or NB:BP or the K_(d) of the parental AB towardsthe target. Conversely, the binding affinity of the NB-containingactivatable antibody towards the target is lower than the bindingaffinity of the AB when it is not associated with the NB or NB:BP orlower than the binding affinity of the parental AB towards the target.For example, the binding affinity of the NB-containing activatableantibody toward the target is at least 5, 10, 25, 50, 100, 250, 500,1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100,10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000,10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or100,000-10,000,000 times lower than the binding affinity of the AB whenit is not associated with the NB or NB:BP or lower than the bindingaffinity of the parental AB towards the target.

When the NB-containing activatable antibody is in the presence of thetarget, specific binding of the AB to the target is reduced orinhibited, as compared to the specific binding of the AB when it is notassociated with the NB or NB:BP. When the NB-containing activatableantibody is in the presence of the target, specific binding of the AB tothe target is reduced or inhibited, as compared to the specific bindingof the parental AB to the target. When compared to the binding of the ABnot associated with an NB or NB:BP or the binding of the parental AB tothe target, the ability of the NB-containing activatable antibody tobind the target is reduced, for example, by at least 50%, 60%, 70%, 80%,90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% for at least2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10,15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 months or longer when measured in vitro and/or in vivo.

When the NB-containing activatable antibody is in the presence of thetarget but not in the presence of a modifying agent (for example aprotease or other enzyme), specific binding of the AB to the target isreduced or inhibited, as compared to the specific binding of the AB whenit is not associated with the NB or NB:BP. When the NB-containingactivatable antibody is in the presence of the target but not in thepresence of a modifying agent (for example a protease, other enzyme,reduction agent, or light), specific binding of the AB to the target isreduced or inhibited, as compared to the specific binding of theparental AB to the target. When compared to the binding of the AB notassociated with an NB or NB:BP or the binding of the parental AB to thetarget, the ability of the NB-containing activatable antibody to bindthe target is reduced, for example, by at least 50%, 60%, 70%, 80%, 90%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% for at least 2, 4,6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30,45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 months or longer when measured in vitro and/or in vivo.

In some examples of any of these activatable antibody embodiments, theactivatable antibody includes an agent conjugated to the AB to producean activatable antibody conjugate. In some embodiments of theactivatable antibody conjugate, the agent is a therapeutic agent. Insome embodiments, the agent is a diagnostic agent. In some embodiments,the agent is a detectable marker. In some embodiments of the activatableantibody conjugate, the agent is an antineoplastic agent. In someembodiments of the activatable antibody conjugate, the agent is a toxinor fragment thereof. In some embodiments of the activatable antibodyconjugate, the agent is conjugated to the AB via a linker. In someembodiments of the activatable antibody conjugate, the linker is acleavable linker. In some embodiments, the agent is conjugated to the ABvia a noncleavable linker. In some embodiments, the agent is amicrotubule inhibitor. In some embodiments, the agent is a nucleic aciddamaging agent, such as a DNA alkylator or DNA intercalator, or otherDNA damaging agent. In some embodiments, the agent is an agent selectedfrom the group listed in Table 3. In some embodiments, the agent is adolastatin. In some embodiments, the agent is an auristatin orderivative thereof. In some embodiments, the agent is auristatin E or aderivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid ormaytansinoid derivative. In some embodiments, the agent is DM1 or DM4.In some embodiments, the agent is a duocarmycin or derivative thereof.In some embodiments, the agent is a calicheamicin or derivative thereof.In some embodiments, the agent is a pyrrolobenzodiazepine.

In some examples of any of these activatable antibody embodiments, theactivatable antibodies are dual-target binding activatable antibodies.Such dual target binding activatable antibodies contain two Abs that maybind the same or different targets. In specific embodiments,dual-targeting activatable antibodies contain bispecific antibodies orantibody fragments.

Dual target binding activatable antibodies are designed so as to have aCL cleavable by a cleaving agent that is co-localized in a target tissuewith one or both of the targets capable of binding to the ABs of theactivatable antibodies. Dual target binding activatable antibodies withmore than one AB to the same or different targets can be designed so asto have more than one CL, wherein the first CL is cleavable by acleaving agent in a first target tissue and wherein the second CL iscleavable by a cleaving agent in a second target tissue, with one ormore of the targets binding to the ABs of the activatable antibodies. Inone embodiment, the first and second target tissues are spatiallyseparated, for example, at different sites in the organism. In oneembodiment, the first and second target tissues are the same tissuetemporally separated, for example the same tissue at two differentpoints in time, for example the first time point is when the tissue isan early stage tumor, and the second time point is when the tissue is alate stage tumor.

The disclosure also provides nucleic acid molecules encoding theactivatable antibodies described herein. The disclosure also providesvectors that include these nucleic acids. The activatable antibodiesdescribed herein are produced by culturing a cell under conditions thatlead to expression of the activatable antibody, wherein the cellincludes these nucleic acid molecules or vectors.

The disclosure also provides methods of manufacturing activatableantibodies. In one embodiment, the method includes the steps of (a)culturing a cell that includes a nucleic acid construct that encodes theactivatable antibody under conditions that lead to expression of theactivatable antibody, wherein the activatable antibody includes (i) anon-binding steric moiety (NB); (ii) a cleavable linker (CL); and (iii)an antibody or an antigen binding fragment thereof (AB) thatspecifically binds a target, wherein (1) the NB does not bindspecifically to the AB; (2) the CL is a polypeptide that includes asubstrate (S) for an enzyme; (3) the CL is positioned such that in anuncleaved state, the NB interferes with binding of the AB to the targetand in a cleaved state, the NB does not interfere with binding of the ABto the target; and (4) the NB does not inhibit cleavage of the CL by theenzyme; and (b) recovering the activatable antibody.

In some embodiments, the method includes the steps of (a) culturing acell that includes a nucleic acid construct that encodes the activatableantibody under conditions that lead to expression of the activatableantibody, wherein the activatable antibody includes (i) a bindingpartner (BP) for a non-binding steric moiety (NB); (ii) a cleavablelinker (CL); and (iii) an antibody or an antigen binding fragmentthereof (AB) that specifically binds a target, wherein (1) the NB doesnot bind specifically to the AB; (2) the CL is a polypeptide thatincludes a substrate (S) for an enzyme; (3) the CL is positioned suchthat in an uncleaved state in the presence of the NB, the NB interfereswith binding of the AB to the target and in a cleaved state, the NB doesnot interfere with binding of the AB to the target and the BP does notinterfere with binding of the AB to the target; and (4) the NB and theBP do not inhibit cleavage of the CL by the enzyme; and (b) recoveringthe activatable antibody. In some examples of this embodiment, the BP ofthe activatable antibody is bound to the NB.

Use of Activatable Antibodies and Conjugated Activatable Antibodies

It will be appreciated that administration of therapeutic entities inaccordance with the disclosure will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present disclosure, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Therapeutic formulations of the disclosure, which include a conjugatedantibody, an activatable antibody and/or a conjugated activatableantibody, are used to prevent, treat or otherwise ameliorate a diseaseor disorder associated with aberrant target expression and/or activity.For example, therapeutic formulations of the disclosure, which include aconjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody, are used to treat or otherwise ameliorateinflammation, an inflammatory disorder, an autoimmune disease and/or acancer or other neoplastic condition. In some embodiments, the cancer isa solid tumor or a hematologic malignancy where the target is expressed.In some embodiments, the cancer is a solid tumor where the target isexpressed. In some embodiments, the cancer is a hematologic malignancywhere the target is expressed. In some embodiments, the target isexpressed on parenchyma (e.g., in cancer, the portion of an organ ortissue that often carries out function(s) of the organ or tissue). Insome embodiments, the target is expressed on a cell, tissue, or organ.In some embodiments, the target is expressed on stroma (i.e., theconnective supportive framework of a cell, tissue, or organ). In someembodiments, the target is expressed on an osteoblast. In someembodiments, the target is expressed on the endothelium (vasculature).In some embodiments, the target is expressed on a cancer stem cell. Insome embodiments, the agent to which the activatable antibody isconjugated is a microtubule inhibitor. In some embodiments, the agent towhich the activatable antibody is conjugated is a nucleic acid damagingagent.

Efficaciousness of prevention, amelioration or treatment is determinedin association with any known method for diagnosing or treating thedisease or disorder associated with target expression and/or activity,such as, for example, aberrant target expression and/or activity.Prolonging the survival of a subject or otherwise delaying theprogression of the disease or disorder associated with target expressionand/or activity, e.g., aberrant target expression and/or activity, in asubject indicates that the conjugated antibody, activatable antibodyand/or conjugated activatable antibody confers a clinical benefit.

A conjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody can be administered in the form of pharmaceuticalcompositions. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, for example, in Remington: The Science And Practice OfPharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co.,Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities,Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa.,1994; and Peptide And Protein Drug Delivery (Advances In ParenteralSciences, Vol. 4), 1991, M. Dekker, New York.

In some embodiments where antibody fragments are used, the smallestfragment that specifically binds to the binding domain of the targetprotein is selected. For example, based upon the variable-regionsequences of an antibody, peptide molecules can be designed that retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology.(See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893(1993)). The formulation can also contain more than one active compoundsas necessary for the particular indication being treated, for example,in some embodiments, those with complementary activities that do notadversely affect each other. In some embodiments, or in addition, thecomposition can comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

In some embodiments, the conjugated antibody, activatable antibodyand/or conjugated activatable antibody contains a detectable label. Anintact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)₂) isused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently-labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently-labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. Included within theusage of the term “biological sample”, therefore, is blood and afraction or component of blood including blood serum, blood plasma, orlymph. That is, the detection method of the disclosure can be used todetect an analyte mRNA, protein, or genomic DNA in a biological samplein vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of an analyte proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, immunochemical staining, and immunofluorescence.In vitro techniques for detection of an analyte genomic DNA includeSouthern hybridizations. Procedures for conducting immunoassays aredescribed, for example in “ELISA: Theory and Practice: Methods inMolecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa,N.J., 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, AcademicPress, Inc., San Diego, Calif., 1996; and “Practice and Theory of EnzymeImmunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Furthermore, in vivo techniques for detection of an analyte proteininclude introducing into a subject a labeled anti-analyte proteinantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

The conjugated antibodies, activatable antibodies and/or conjugatedactivatable antibodies of the disclosure are also useful in a variety ofdiagnostic and prophylactic formulations. In one embodiment, aconjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody is administered to patients that are at risk ofdeveloping one or more of the aforementioned disorders. A patient's ororgan's predisposition to one or more of the aforementioned disorderscan be determined using genotypic, serological or biochemical markers.

In some embodiments, a conjugated antibody, an activatable antibodyand/or a conjugated activatable antibody is administered to humanindividuals diagnosed with a clinical indication associated with one ormore of the aforementioned disorders. Upon diagnosis, a conjugatedantibody, an activatable antibody and/or a conjugated activatableantibody is administered to mitigate or reverse the effects of theclinical indication.

A conjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody of the disclosure is also useful in the detectionof a target in patient samples and accordingly are useful asdiagnostics. For example, the antibodies and/or activatable antibodies,and conjugated versions thereof, of the disclosure are used in in vitroassays, e.g., ELISA, to detect target levels in a patient sample.

In one embodiment, a conjugated antibody, an activatable antibody and/ora conjugated activatable antibody of the disclosure is immobilized on asolid support (e.g., the well(s) of a microtiter plate). The immobilizedconjugated antibody, activatable antibody and/or conjugated activatableantibody serves as a capture antibody for any target that may be presentin a test sample. Prior to contacting the immobilized antibody with apatient sample, the solid support is rinsed and treated with a blockingagent such as milk protein or albumin to prevent nonspecific adsorptionof the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of target antigen in the test sample is determined bycomparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using theantibodies of the disclosure, and conjugated versions thereof, in an invitro diagnostic assay, it is possible to stage a disease in a subjectbased on expression levels of the target antigen. For a given disease,samples of blood are taken from subjects diagnosed as being at variousstages in the progression of the disease, and/or at various points inthe therapeutic treatment of the disease. Using a population of samplesthat provides statistically significant results for each stage ofprogression or therapy, a range of concentrations of the antigen thatmay be considered characteristic of each stage is designated.

A conjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody can also be used in diagnostic and/or imagingmethods. In some embodiments, such methods are in vitro methods. In someembodiments, such methods are in vivo methods. In some embodiments, suchmethods are in situ methods. In some embodiments, such methods are exvivo methods. For example, activatable antibodies having anenzymatically cleavable CM can be used to detect the presence or absenceof an enzyme that is capable of cleaving the CM. Such activatableantibodies can be used in diagnostics, which can include in vivodetection (e.g., qualitative or quantitative) of enzyme activity (or, insome embodiments, an environment of increased reduction potential suchas that which can provide for reduction of a disulfide bond) throughmeasured accumulation of activated antibodies (i.e., antibodiesresulting from cleavage of an activatable antibody) in a given cell ortissue of a given host organism. Such accumulation of activatedantibodies indicates not only that the tissue expresses enzymaticactivity (or an increased reduction potential depending on the nature ofthe CM) but also that the tissue expresses target to which the activatedantibody binds.

For example, the CM can be selected to be substrate for a matrixmetalloprotease (MMP) found at the site of a tumor, at the site of aviral or bacterial infection at a biologically confined site (e.g., suchas in an abscess, in an organ, and the like), and the like. The AB canbe one that binds a target antigen. Using methods as disclosed herein,or when appropriate, methods familiar to one skilled in the art, adetectable label (e.g., a fluorescent label or radioactive label orradiotracer) can be conjugated to an AB or other region of an antibodyand/or activatable antibody. Suitable detectable labels are discussed inthe context of the above screening methods and additional specificexamples are provided below. Using an AB specific to a protein orpeptide of the disease state, along with an MMP whose activity iselevated in the disease tissue of interest, activatable antibodies willexhibit an increased rate of binding to disease tissue relative totissues where the CM specific enzyme is not present at a detectablelevel or is present at a lower level than in disease tissue or isinactive (e.g., in zymogen form or in complex with an inhibitor). Sincesmall proteins and peptides are rapidly cleared from the blood by therenal filtration system, and because the enzyme specific for the CM isnot present at a detectable level (or is present at lower levels innon-disease tissues or is present in inactive conformation),accumulation of activated antibodies in the disease tissue is enhancedrelative to non-disease tissues.

In another example, activatable antibodies can be used to detect thepresence or absence of a cleaving agent in a sample. For example, wherethe activatable antibodies contain a CM susceptible to cleavage by anenzyme, the activatable antibodies can be used to detect (eitherqualitatively or quantitatively) the presence of an enzyme in thesample. In another example, where the activatable antibodies contain aCM susceptible to cleavage by reducing agent, the activatable antibodiescan be used to detect (either qualitatively or quantitatively) thepresence of reducing conditions in a sample. To facilitate analysis inthese methods, the activatable antibodies can be detectably labeled, andcan be bound to a support (e.g., a solid support, such as a slide orbead). The detectable label can be positioned on a portion of theactivatable antibody that is not released following cleavage, forexample, the detectable label can be a quenched fluorescent label orother label that is not detectable until cleavage has occurred. Theassay can be conducted by, for example, contacting the immobilized,detectably labeled activatable antibodies with a sample suspected ofcontaining an enzyme and/or reducing agent for a time sufficient forcleavage to occur, then washing to remove excess sample andcontaminants. The presence or absence of the cleaving agent (e.g.,enzyme or reducing agent) in the sample is then assessed by a change indetectable signal of the activatable antibodies prior to contacting withthe sample e.g., the presence of and/or an increase in detectable signaldue to cleavage of the activatable antibody by the cleaving agent in thesample.

Such detection methods can be adapted to also provide for detection ofthe presence or absence of a target that is capable of binding the AB ofthe activatable antibodies when cleaved. Thus, the assays can be adaptedto assess the presence or absence of a cleaving agent and the presenceor absence of a target of interest. The presence or absence of thecleaving agent can be detected by the presence of and/or an increase indetectable label of the activatable antibodies as described above, andthe presence or absence of the target can be detected by detection of atarget-AB complex e.g., by use of a detectably labeled anti-targetantibody.

Activatable antibodies are also useful in in situ imaging for thevalidation of activatable antibody activation, e.g., by proteasecleavage, and binding to a particular target. In situ imaging is atechnique that enables localization of proteolytic activity and targetin biological samples such as cell cultures or tissue sections. Usingthis technique, it is possible to confirm both binding to a given targetand proteolytic activity based on the presence of a detectable label(e.g., a fluorescent label).

These techniques are useful with any frozen cells or tissue derived froma disease site (e.g. tumor tissue) or healthy tissues. These techniquesare also useful with fresh cell or tissue samples.

In these techniques, an activatable antibody is labeled with adetectable label. The detectable label may be a fluorescent dye, (e.g. afluorophore, Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate(TRITC), an Alexa Fluor® label), a near infrared (NIR) dye (e.g., Qdot®nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotinand an amplification reagent such as streptavidin, or an enzyme (e.g.horseradish peroxidase or alkaline phosphatase).

Detection of the label in a sample that has been incubated with thelabeled, activatable antibody indicates that the sample contains thetarget and contains a matrix metalloprotease (MMP) that is specific forthe CM of the activatable antibody. In some embodiments, the presence ofthe MMP can be confirmed using broad spectrum protease inhibitors suchas those described herein, and/or by using an agent that is specific forthe protease, for example, an antibody such as A11, which is specificfor the protease matriptase (MT-SP1) and inhibits the proteolyticactivity of matriptase; see e.g., International Publication Number WO2010/129609, published 11 Nov. 2010. The same approach of using broadspectrum protease inhibitors such as those described herein, and/or byusing a more selective inhibitory agent can be used to identify a MMPspecific for the CM of the activatable antibody. In some embodiments,the presence of the target can be confirmed using an agent that isspecific for the target, e.g., another antibody, or the detectable labelcan be competed with unlabeled target. In some embodiments, unlabeledactivatable antibody could be used, with detection by a labeledsecondary antibody or more complex detection system.

Similar techniques are also useful for in vivo imaging where detectionof the fluorescent signal in a subject, e.g., a mammal, including ahuman, indicates that the disease site contains the target and containsa MMP that is specific for the CM of the activatable antibody.

These techniques are also useful in kits and/or as reagents for thedetection, identification or characterization of protease activity in avariety of cells, tissues, and organisms based on the protease-specificCM in the activatable antibody.

The disclosure provides methods of using the antibodies and/oractivatable antibodies in a variety of diagnostic and/or prophylacticindications. For example, the disclosure provides methods of detectingpresence or absence of a cleaving agent and a target of interest in asubject or a sample by (i) contacting a subject or sample with anactivatable antibody, wherein the activatable antibody comprises amasking moiety (MM), a cleavable moiety (CM) that is cleaved by thecleaving agent, and an antigen binding domain or fragment thereof (AB)that specifically binds the target of interest, wherein the activatableantibody in an uncleaved, non-activated state comprises a structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of theAB to the target, and wherein the MM does not have an amino acidsequence of a naturally occurring binding partner of the AB and is not amodified form of a natural binding partner of the AB; and (b) wherein,in an uncleaved, non-activated state, the MM interferes with specificbinding of the AB to the target, and in a cleaved, activated state theMM does not interfere or compete with specific binding of the AB to thetarget; and (ii) measuring a level of activated activatable antibody inthe subject or sample, wherein a detectable level of activatedactivatable antibody in the subject or sample indicates that thecleaving agent and the target are present in the subject or sample andwherein no detectable level of activated activatable antibody in thesubject or sample indicates that the cleaving agent, the target or boththe cleaving agent and the target are absent and/or not sufficientlypresent in the subject or sample. In some embodiments, the activatableantibody is an activatable antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable antibody is notconjugated to an agent. In some embodiments, the activatable antibodycomprises a detectable label. In some embodiments, the detectable labelis positioned on the AB. In some embodiments, measuring the level ofactivatable antibody in the subject or sample is accomplished using asecondary reagent that specifically binds to the activated antibody,wherein the reagent comprises a detectable label. In some embodiments,the secondary reagent is an antibody comprising a detectable label.

The disclosure also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with an activatable antibody in the presence of a target ofinterest, e.g., the target, wherein the activatable antibody comprises amasking moiety (MM), a cleavable moiety (CM) that is cleaved by thecleaving agent, and an antigen binding domain or fragment thereof (AB)that specifically binds the target of interest, wherein the activatableantibody in an uncleaved, non-activated state comprises a structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of theAB to the target, and wherein the MM does not have an amino acidsequence of a naturally occurring binding partner of the AB and is not amodified form of a natural binding partner of the AB; and (b) wherein,in an uncleaved, non-activated state, the MM interferes with specificbinding of the AB to the target, and in a cleaved, activated state theMM does not interfere or compete with specific binding of the AB to thetarget; and (ii) measuring a level of activated activatable antibody inthe subject or sample, wherein a detectable level of activatedactivatable antibody in the subject or sample indicates that thecleaving agent is present in the subject or sample and wherein nodetectable level of activated activatable antibody in the subject orsample indicates that the cleaving agent is absent and/or notsufficiently present in the subject or sample. In some embodiments, theactivatable antibody is an activatable antibody to which a therapeuticagent is conjugated. In some embodiments, the activatable antibody isnot conjugated to an agent. In some embodiments, the activatableantibody comprises a detectable label. In some embodiments, thedetectable label is positioned on the AB. In some embodiments, measuringthe level of activatable antibody in the subject or sample isaccomplished using a secondary reagent that specifically binds to theactivated antibody, wherein the reagent comprises a detectable label. Insome embodiments, the secondary reagent is an antibody comprising adetectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent and the target in a subject or asample, where the kits include at least an activatable antibodycomprises a masking moiety (MM), a cleavable moiety (CM) that is cleavedby the cleaving agent, and an antigen binding domain or fragment thereof(AB) that specifically binds the target of interest, wherein theactivatable antibody in an uncleaved, non-activated state comprises astructural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibitsbinding of the AB to the target, and wherein the MM does not have anamino acid sequence of a naturally occurring binding partner of the ABand is not a modified form of a natural binding partner of the AB; and(b) wherein, in an uncleaved, non-activated state, the MM interfereswith specific binding of the AB to the target, and in a cleaved,activated state the MM does not interfere or compete with specificbinding of the AB to the target; and (ii) measuring a level of activatedactivatable antibody in the subject or sample, wherein a detectablelevel of activated activatable antibody in the subject or sampleindicates that the cleaving agent is present in the subject or sampleand wherein no detectable level of activated activatable antibody in thesubject or sample indicates that the cleaving agent is absent and/or notsufficiently present in the subject or sample. In some embodiments, theactivatable antibody is an activatable antibody to which a therapeuticagent is conjugated. In some embodiments, the activatable antibody isnot conjugated to an agent. In some embodiments, the activatableantibody comprises a detectable label. In some embodiments, thedetectable label is positioned on the AB. In some embodiments, measuringthe level of activatable antibody in the subject or sample isaccomplished using a secondary reagent that specifically binds to theactivated antibody, wherein the reagent comprises a detectable label. Insome embodiments, the secondary reagent is an antibody comprising adetectable label.

The disclosure also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with an activatable antibody, wherein the activatable antibodycomprises a masking moiety (MM), a cleavable moiety (CM) that is cleavedby the cleaving agent, an antigen binding domain (AB) that specificallybinds the target, and a detectable label, wherein the activatableantibody in an uncleaved, non-activated state comprises a structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM; wherein the MM is a peptide that inhibits binding of the AB tothe target, and wherein the MM does not have an amino acid sequence of anaturally occurring binding partner of the AB and is not a modified formof a natural binding partner of the AB; wherein, in an uncleaved,non-activated state, the MM interferes with specific binding of the ABto the target, and in a cleaved, activated state the MM does notinterfere or compete with specific binding of the AB to the target; andwherein the detectable label is positioned on a portion of theactivatable antibody that is released following cleavage of the CM; and(ii) measuring a level of detectable label in the subject or sample,wherein a detectable level of the detectable label in the subject orsample indicates that the cleaving agent is absent and/or notsufficiently present in the subject or sample and wherein no detectablelevel of the detectable label in the subject or sample indicates thatthe cleaving agent is present in the subject or sample. In someembodiments, the activatable antibody is an activatable antibody towhich a therapeutic agent is conjugated. In some embodiments, theactivatable antibody is not conjugated to an agent. In some embodiments,the activatable antibody comprises a detectable label. In someembodiments, the detectable label is positioned on the AB. In someembodiments, measuring the level of activatable antibody in the subjector sample is accomplished using a secondary reagent that specificallybinds to the activated antibody, wherein the reagent comprises adetectable label. In some embodiments, the secondary reagent is anantibody comprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent and the target in a subject or asample, where the kits include at least an activatable antibody and/orconjugated activatable antibody (e.g., an activatable antibody to whicha therapeutic agent is conjugated) described herein for use incontacting a subject or biological sample and means for detecting thelevel of activated activatable antibody and/or conjugated activatableantibody in the subject or biological sample, wherein a detectable levelof activated activatable antibody in the subject or biological sampleindicates that the cleaving agent and the target are present in thesubject or biological sample and wherein no detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent, the target or both the cleaving agentand the target are absent and/or not sufficiently present in the subjector biological sample, such that the target binding and/or proteasecleavage of the activatable antibody cannot be detected in the subjector biological sample.

The disclosure also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orbiological sample with an activatable antibody in the presence of thetarget, and (ii) measuring a level of activated activatable antibody inthe subject or biological sample, wherein a detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent is present in the subject orbiological sample and wherein no detectable level of activatedactivatable antibody in the subject or biological sample indicates thatthe cleaving agent is absent and/or not sufficiently present in thesubject or biological sample at a detectable level, such that proteasecleavage of the activatable antibody cannot be detected in the subjector biological sample. Such an activatable antibody includes a maskingmoiety (MM), a cleavable moiety (CM) that is cleaved by the cleavingagent, and an antigen binding domain or fragment thereof (AB) thatspecifically binds the target, wherein the activatable antibody in anuncleaved (i.e., non-activated) state comprises a structural arrangementfrom N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)wherein the MM is a peptide that inhibits binding of the AB to thetarget, and wherein the MM does not have an amino acid sequence of anaturally occurring binding partner of the AB; and (b) wherein the MM ofthe activatable antibody in an uncleaved state interferes with specificbinding of the AB to the target, and wherein the MM of an activatableantibody in a cleaved (i.e., activated) state does not interfere orcompete with specific binding of the AB to the target. In someembodiments, the activatable antibody is an activatable antibody towhich a therapeutic agent is conjugated. In some embodiments, theactivatable antibody is not conjugated to an agent. In some embodiments,the detectable label is attached to the masking moiety. In someembodiments, the detectable label is attached to the cleavable moietyN-terminal to the protease cleavage site. In some embodiments, a singleantigen binding site of the AB is masked. In some embodiments wherein anantibody of the disclosure has at least two antigen binding sites, atleast one antigen binding site is masked and at least one antigenbinding site is not masked. In some embodiments, all antigen bindingsites are masked. In some embodiments, the measuring step includes useof a secondary reagent comprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent and the target in a subject or asample, where the kits include at least an activatable antibody and/orconjugated activatable antibody described herein for use in contacting asubject or biological sample with an activatable antibody in thepresence of the target, and measuring a level of activated activatableantibody in the subject or biological sample, wherein a detectable levelof activated activatable antibody in the subject or biological sampleindicates that the cleaving agent is present in the subject orbiological sample and wherein no detectable level of activatedactivatable antibody in the subject or biological sample indicates thatthe cleaving agent is absent and/or not sufficiently present in thesubject or biological sample at a detectable level, such that proteasecleavage of the activatable antibody cannot be detected in the subjector biological sample. Such an activatable antibody includes a maskingmoiety (MM), a cleavable moiety (CM) that is cleaved by the cleavingagent, and an antigen binding domain or fragment thereof (AB) thatspecifically binds the target, wherein the activatable antibody in anuncleaved (i.e., non-activated) state comprises a structural arrangementfrom N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)wherein the MM is a peptide that inhibits binding of the AB to thetarget, and wherein the MM does not have an amino acid sequence of anaturally occurring binding partner of the AB; and (b) wherein the MM ofthe activatable antibody in an uncleaved state interferes with specificbinding of the AB to the target, and wherein the MM of an activatableantibody in a cleaved (i.e., activated) state does not interfere orcompete with specific binding of the AB to the target. In someembodiments, the activatable antibody is an activatable antibody towhich a therapeutic agent is conjugated. In some embodiments, theactivatable antibody is not conjugated to an agent. In some embodiments,the detectable label is attached to the masking moiety. In someembodiments, the detectable label is attached to the cleavable moietyN-terminal to the protease cleavage site. In some embodiments, a singleantigen binding site of the AB is masked. In some embodiments wherein anantibody of the disclosure has at least two antigen binding sites, atleast one antigen binding site is masked and at least one antigenbinding site is not masked. In some embodiments, all antigen bindingsites are masked. In some embodiments, the measuring step includes useof a secondary reagent comprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent in a subject or a sample, wherethe kits include at least an activatable antibody and/or conjugatedactivatable antibody described herein for use in contacting a subject orbiological sample and means for detecting the level of activatedactivatable antibody and/or conjugated activatable antibody in thesubject or biological sample, wherein the activatable antibody includesa detectable label that is positioned on a portion of the activatableantibody that is released following cleavage of the CM, wherein adetectable level of activated activatable antibody in the subject orbiological sample indicates that the cleaving agent is absent and/or notsufficiently present in the subject or biological sample such that thetarget binding and/or protease cleavage of the activatable antibodycannot be detected in the subject or biological sample, and wherein nodetectable level of activated activatable antibody in the subject orbiological sample indicates that the cleaving agent is present in thesubject or biological sample at a detectable level.

The disclosure provides methods of detecting presence or absence of acleaving agent and the target in a subject or a sample by (i) contactinga subject or biological sample with an activatable antibody, wherein theactivatable antibody includes a detectable label that is positioned on aportion of the activatable antibody that is released following cleavageof the CM and (ii) measuring a level of activated activatable antibodyin the subject or biological sample, wherein a detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent, the target or both the cleaving agentand the target are absent and/or not sufficiently present in the subjector biological sample, such that the target binding and/or proteasecleavage of the activatable antibody cannot be detected in the subjector biological sample, and wherein a reduced detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent and the target are present in thesubject or biological sample. A reduced level of detectable label is,for example, a reduction of about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95% and/or about 100%. Such an activatable antibodyincludes a masking moiety (MM), a cleavable moiety (CM) that is cleavedby the cleaving agent, and an antigen binding domain or fragment thereof(AB) that specifically binds the target, wherein the activatableantibody in an uncleaved (i.e., non-activated) state comprises astructural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibitsbinding of the AB to the target, and wherein the MM does not have anamino acid sequence of a naturally occurring binding partner of the AB;and (b) wherein the MM of the activatable antibody in an uncleaved stateinterferes with specific binding of the AB to the target, and whereinthe MM of an activatable antibody in a cleaved (i.e., activated) statedoes not interfere or compete with specific binding of the AB to thetarget. In some embodiments, the activatable antibody is an activatableantibody to which a therapeutic agent is conjugated. In someembodiments, the activatable antibody is not conjugated to an agent. Insome embodiments, the activatable antibody comprises a detectable label.In some embodiments, the detectable label is positioned on the AB. Insome embodiments, measuring the level of activatable antibody in thesubject or sample is accomplished using a secondary reagent thatspecifically binds to the activated antibody, wherein the reagentcomprises a detectable label. In some embodiments, the secondary reagentis an antibody comprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent and the target in a subject or asample, where the kits include at least an activatable antibody and/orconjugated activatable antibody described herein for use in contacting asubject or biological sample and means for detecting the level ofactivated activatable antibody and/or conjugated activatable antibody inthe subject or biological sample, wherein a detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent, the target or both the cleaving agentand the target are absent and/or not sufficiently present in the subjector biological sample, such that the target binding and/or proteasecleavage of the activatable antibody cannot be detected in the subjector biological sample, and wherein a reduced detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent and the target are present in thesubject or biological sample. A reduced level of detectable label is,for example, a reduction of about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95% and/or about 100%.

The disclosure also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orbiological sample with an activatable antibody, wherein the activatableantibody includes a detectable label that is positioned on a portion ofthe activatable antibody that is released following cleavage of the CM;and (ii) measuring a level of detectable label in the subject orbiological sample, wherein a detectable level of the detectable label inthe subject or biological sample indicates that the cleaving agent isabsent and/or not sufficiently present in the subject or biologicalsample at a detectable level, such that protease cleavage of theactivatable antibody cannot be detected in the subject or biologicalsample, and wherein a reduced detectable level of the detectable labelin the subject or biological sample indicates that the cleaving agent ispresent in the subject or biological sample. A reduced level ofdetectable label is, for example, a reduction of about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95% and/or about 100%. Such anactivatable antibody includes a masking moiety (MM), a cleavable moiety(CM) that is cleaved by the cleaving agent, and an antigen bindingdomain or fragment thereof (AB) that specifically binds the target,wherein the activatable antibody in an uncleaved (i.e., non-activated)state comprises a structural arrangement from N-terminus to C-terminusas follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide thatinhibits binding of the AB to the target, and wherein the MM does nothave an amino acid sequence of a naturally occurring binding partner ofthe AB; and (b) wherein the MM of the activatable antibody in anuncleaved state interferes with specific binding of the AB to thetarget, and wherein the MM of an activatable antibody in a cleaved(i.e., activated) state does not interfere or compete with specificbinding of the AB to the target. In some embodiments, the activatableantibody is an activatable antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable antibody is notconjugated to an agent. In some embodiments, the activatable antibodycomprises a detectable label. In some embodiments, the detectable labelis positioned on the AB. In some embodiments, measuring the level ofactivatable antibody in the subject or sample is accomplished using asecondary reagent that specifically binds to the activated antibody,wherein the reagent comprises a detectable label. In some embodiments,the secondary reagent is an antibody comprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent of interest in a subject or asample, where the kits include at least an activatable antibody and/orconjugated activatable antibody described herein for use in contacting asubject or biological sample and means for detecting the level ofactivated activatable antibody and/or conjugated activatable antibody inthe subject or biological sample, wherein the activatable antibodyincludes a detectable label that is positioned on a portion of theactivatable antibody that is released following cleavage of the CM,wherein a detectable level of the detectable label in the subject orbiological sample indicates that the cleaving agent, the target, or boththe cleaving agent and the target are absent and/or not sufficientlypresent in the subject or biological sample, such that the targetbinding and/or protease cleavage of the activatable antibody cannot bedetected in the subject or biological sample, and wherein a reduceddetectable level of the detectable label in the subject or biologicalsample indicates that the cleaving agent and the target are present inthe subject or biological sample. A reduced level of detectable labelis, for example, a reduction of about 5%, about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95% and/or about 100%.

In some embodiments of these methods and kits, the activatable antibodyincludes a detectable label. In some embodiments of these methods andkits, the detectable label includes an imaging agent, a contrastingagent, an enzyme, a fluorescent label, a chromophore, a dye, one or moremetal ions, or a ligand-based label. In some embodiments of thesemethods and kits, the imaging agent comprises a radioisotope. In someembodiments of these methods and kits, the radioisotope is indium ortechnetium. In some embodiments of these methods and kits, thecontrasting agent comprises iodine, gadolinium or iron oxide. In someembodiments of these methods and kits, the enzyme comprises horseradishperoxidase, alkaline phosphatase, or β-galactosidase. In someembodiments of these methods and kits, the fluorescent label comprisesyellow fluorescent protein (YFP), cyan fluorescent protein (CFP), greenfluorescent protein (GFP), modified red fluorescent protein (mRFP), redfluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europiumderivative. In some embodiments of these methods and kits, theluminescent label comprises an N-methylacrydium derivative. In someembodiments of these methods, the label comprises an Alexa Fluor® label,such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments ofthese methods and kits, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

In some embodiments of these methods and kits, the subject is a mammal.In some embodiments of these methods and kits, the subject is a human.In some embodiments, the subject is a non-human mammal, such as anon-human primate, companion animal (e.g., cat, dog, horse), farmanimal, work animal, or zoo animal. In some embodiments, the subject isa rodent.

In some embodiments of these methods, the method is an in vivo method.In some embodiments of these methods, the method is an in situ method.In some embodiments of these methods, the method is an ex vivo method.In some embodiments of these methods, the method is an in vitro method.

In some embodiments, in situ imaging and/or in vivo imaging are usefulin methods to identify which patients to treat. For example, in in situimaging, the activatable antibodies are used to screen patient samplesto identify those patients having the appropriate protease(s) andtarget(s) at the appropriate location, e.g., at a tumor site.

In some embodiments, in situ imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableantibody of the disclosure. For example, patients that test positive forboth the target (e.g., the target) and a protease that cleaves thesubstrate in the cleavable moiety (CM) of the activatable antibody beingtested (e.g., accumulate activated antibodies at the disease site) areidentified as suitable candidates for treatment with such an activatableantibody comprising such a CM. Likewise, patients that test negative foreither or both of the target (e.g., the target) and the protease thatcleaves the substrate in the CM in the activatable antibody being testedusing these methods might be identified as suitable candidates foranother form of therapy. In some embodiments, such patients that testnegative with respect to a first activatable antibody can be tested withother activatable antibodies comprising different CMs until a suitableactivatable antibody for treatment is identified (e.g., an activatableantibody comprising a CM that is cleaved by the patient at the site ofdisease). In some embodiments, the patient is then administered atherapeutically effective amount of the conjugated activatable antibodyfor which the patient tested positive.

In some embodiments, in vivo imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableantibody of the disclosure. For example, patients that test positive forboth the target (e.g., the target) and a protease that cleaves thesubstrate in the cleavable moiety (CM) of the activatable antibody beingtested (e.g., accumulate activated antibodies at the disease site) areidentified as suitable candidates for treatment with such an activatableantibody comprising such a CM. Likewise, patients that test negativemight be identified as suitable candidates for another form of therapy.In some embodiments, such patients that test negative with respect to afirst activatable antibody can be tested with other activatableantibodies comprising different CMs until a suitable activatableantibody for treatment is identified (e.g., an activatable antibodycomprising a CM that is cleaved by the patient at the site of disease).In some embodiments, the patient is then administered a therapeuticallyeffective amount of the conjugated activatable antibody for which thepatient tested positive.

In some embodiments of the methods and kits, the method or kit is usedto identify or otherwise refine a patient population suitable fortreatment with an activatable antibody of the disclosure. For example,patients that test positive for both the target (e.g., the target) and aprotease that cleaves the substrate in the cleavable moiety (CM) of theactivatable antibody being tested in these methods are identified assuitable candidates for treatment with such an activatable antibodycomprising such a CM. Likewise, patients that test negative for both ofthe targets (e.g., the target) and the protease that cleaves thesubstrate in the CM in the activatable antibody being tested using thesemethods might be identified as suitable candidates for another form oftherapy. In some embodiments, such patients can be tested with otheractivatable antibodies until a suitable activatable antibody fortreatment is identified (e.g., an activatable antibody comprising a CMthat is cleaved by the patient at the site of disease). In someembodiments, patients that test negative for either of the target (e.g.,the target) are identified as suitable candidates for treatment withsuch an activatable antibody comprising such a CM. In some embodiments,patients that test negative for either of the target (e.g., the target)are identified as not being suitable candidates for treatment with suchan activatable antibody comprising such a CM. In some embodiments, suchpatients can be tested with other activatable antibodies until asuitable activatable antibody for treatment is identified (e.g., anactivatable antibody comprising a CM that is cleaved by the patient atthe site of disease). In some embodiments, the activatable antibody isan activatable antibody to which a therapeutic agent is conjugated. Insome embodiments, the activatable antibody is not conjugated to anagent. In some embodiments, the activatable antibody comprises adetectable label. In some embodiments, the detectable label ispositioned on the AB. In some embodiments, measuring the level ofactivatable antibody in the subject or sample is accomplished using asecondary reagent that specifically binds to the activated antibody,wherein the reagent comprises a detectable label. In some embodiments,the secondary reagent is an antibody comprising a detectable label.

In some embodiments, a method or kit is used to identify or otherwiserefine a patient population suitable for treatment with an anti-thetarget activatable antibody and/or conjugated activatable antibody(e.g., activatable antibody to which a therapeutic agent is conjugated)of the disclosure, followed by treatment by administering thatactivatable antibody and/or conjugated activatable antibody to a subjectin need thereof. For example, patients that test positive for both thetargets (e.g., the target) and a protease that cleaves the substrate inthe cleavable moiety (CM) of the activatable antibody and/or conjugatedactivatable antibody being tested in these methods are identified assuitable candidates for treatment with such antibody and/or such aconjugated activatable antibody comprising such a CM, and the patient isthen administered a therapeutically effective amount of the activatableantibody and/or conjugated activatable antibody that was tested.Likewise, patients that test negative for either or both of the target(e.g., the target) and the protease that cleaves the substrate in the CMin the activatable antibody being tested using these methods might beidentified as suitable candidates for another form of therapy. In someembodiments, such patients can be tested with other antibody and/orconjugated activatable antibody until a suitable antibody and/orconjugated activatable antibody for treatment is identified (e.g., anactivatable antibody and/or conjugated activatable antibody comprising aCM that is cleaved by the patient at the site of disease). In someembodiments, the patient is then administered a therapeuticallyeffective amount of the activatable antibody and/or conjugated for whichthe patient tested positive.

In some embodiments of these methods and kits, the MM is a peptidehaving a length from about 4 to 40 amino acids. In some embodiments ofthese methods and kits, the activatable antibody comprises a linkerpeptide, wherein the linker peptide is positioned between the MM and theCM. In some embodiments of these methods and kits, the activatableantibody comprises a linker peptide, where the linker peptide ispositioned between the AB and the CM. In some embodiments of thesemethods and kits, the activatable antibody comprises a first linkerpeptide (L1) and a second linker peptide (L2), wherein the first linkerpeptide is positioned between the MM and the CM and the second linkerpeptide is positioned between the AB and the CM. In some embodiments ofthese methods and kits, each of L1 and L2 is a peptide of about 1 to 20amino acids in length, and wherein each of L1 and L2 need not be thesame linker. In some embodiments of these methods and kits, one or bothof L1 and L2 comprises a glycine-serine polymer. In some embodiments ofthese methods and kits, at least one of L1 and L2 comprises an aminoacid sequence selected from the group consisting of (GS)n, (GSGGS)n (SEQID NO: 1) and (GGGS)n (SEQ ID NO: 2), where n is an integer of at leastone. In some embodiments of these methods and kits, at least one of L1and L2 comprises an amino acid sequence having the formula (GGS)n, wheren is an integer of at least one. In some embodiments of these methodsand kits, at least one of L1 and L2 comprises an amino acid sequenceselected from the group consisting of Gly-Gly-Ser-Gly (SEQ ID NO: 3),Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 4), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 5),Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 7),and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 8).

In some embodiments of these methods and kits, the AB comprises anantibody or antibody fragment sequence selected from the cross-reactiveantibody sequences presented herein. In some embodiments of thesemethods and kits, the AB comprises a Fab fragment, a scFv or a singlechain antibody (scAb).

In some embodiments of these methods and kits, the cleaving agent is aprotease that is co-localized in the subject or sample with the targetand the CM is a polypeptide that functions as a substrate for theprotease, wherein the protease cleaves the CM in the activatableantibody when the activatable antibody is exposed to the protease. Insome embodiments of these methods and kits, the CM is a polypeptide ofup to 15 amino acids in length. In some embodiments of these methods andkits, the CM is coupled to the N-terminus of the AB. In some embodimentsof these methods and kits, the CM is coupled to the C-terminus of theAB. In some embodiments of these methods and kits, the CM is coupled tothe N-terminus of a VL chain of the AB.

The activatable antibodies and/or conjugated activatable antibodies ofthe disclosure are used in diagnostic and prophylactic formulations. Inone embodiment, an activatable antibody is administered to patients thatare at risk of developing one or more of the aforementionedinflammation, inflammatory disorders, cancer or other disorders.

A patient's or organ's predisposition to one or more of theaforementioned disorders can be determined using genotypic, serologicalor biochemical markers.

In some embodiments, an activatable antibody and/or conjugatedactivatable antibodies is administered to human individuals diagnosedwith a clinical indication associated with one or more of theaforementioned disorders. Upon diagnosis, an activatable antibody and/orconjugated activatable antibodies is administered to mitigate or reversethe effects of the clinical indication.

Activatable antibodies and/or conjugated activatable antibodies of thedisclosure are also useful in the detection of the target in patientsamples and accordingly are useful as diagnostics. For example, theactivatable antibodies and/or conjugated activatable antibodies of thedisclosure are used in in vitro assays, e.g., ELISA, to detect targetlevels in a patient sample.

In one embodiment, an activatable antibody of the disclosure isimmobilized on a solid support (e.g., the well(s) of a microtiterplate). The immobilized activatable antibody serves as a captureantibody for any target that may be present in a test sample. Prior tocontacting the immobilized antibody with a patient sample, the solidsupport is rinsed and treated with a blocking agent such as milk proteinor albumin to prevent nonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of target antigen in the test sample is determined bycomparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using theantibodies of the disclosure in an in vitro diagnostic assay, it ispossible to stage a disease in a subject based on expression levels ofthe Target antigen. For a given disease, samples of blood are taken fromsubjects diagnosed as being at various stages in the progression of thedisease, and/or at various points in the therapeutic treatment of thedisease. Using a population of samples that provides statisticallysignificant results for each stage of progression or therapy, a range ofconcentrations of the antigen that may be considered characteristic ofeach stage is designated.

Activatable antibodies and/or conjugated activatable antibodies can alsobe used in diagnostic and/or imaging methods. In some embodiments, suchmethods are in vitro methods. In some embodiments, such methods are invivo methods. In some embodiments, such methods are in situ methods. Insome embodiments, such methods are ex vivo methods. For example,activatable antibodies having an enzymatically cleavable CM can be usedto detect the presence or absence of an enzyme that is capable ofcleaving the CM. Such activatable antibodies can be used in diagnostics,which can include in vivo detection (e.g., qualitative or quantitative)of enzyme activity (or, in some embodiments, an environment of increasedreduction potential such as that which can provide for reduction of adisulfide bond) through measured accumulation of activated antibodies(i.e., antibodies resulting from cleavage of an activatable antibody) ina given cell or tissue of a given host organism. Such accumulation ofactivated antibodies indicates not only that the tissue expressesenzymatic activity (or an increased reduction potential depending on thenature of the CM) but also that the tissue expresses target to which theactivated antibody binds.

For example, the CM can be selected to be a protease substrate for aprotease found at the site of a tumor, at the site of a viral orbacterial infection at a biologically confined site (e.g., such as in anabscess, in an organ, and the like), and the like. The AB can be onethat binds a target antigen. Using methods familiar to one skilled inthe art, a detectable label (e.g., a fluorescent label or radioactivelabel or radiotracer) can be conjugated to an AB or other region of anactivatable antibody. Suitable detectable labels are discussed in thecontext of the above screening methods and additional specific examplesare provided below. Using an AB specific to a protein or peptide of thedisease state, along with a protease whose activity is elevated in thedisease tissue of interest, activatable antibodies will exhibit anincreased rate of binding to disease tissue relative to tissues wherethe CM specific enzyme is not present at a detectable level or ispresent at a lower level than in disease tissue or is inactive (e.g., inzymogen form or in complex with an inhibitor). Since small proteins andpeptides are rapidly cleared from the blood by the renal filtrationsystem, and because the enzyme specific for the CM is not present at adetectable level (or is present at lower levels in non-disease tissuesor is present in inactive conformation), accumulation of activatedantibodies in the disease tissue is enhanced relative to non-diseasetissues.

In another example, activatable antibodies can be used to detect thepresence or absence of a cleaving agent in a sample. For example, wherethe activatable antibodies contain a CM susceptible to cleavage by anenzyme, the activatable antibodies can be used to detect (eitherqualitatively or quantitatively) the presence of an enzyme in thesample. In another example, where the activatable antibodies contain aCM susceptible to cleavage by reducing agent, the activatable antibodiescan be used to detect (either qualitatively or quantitatively) thepresence of reducing conditions in a sample. To facilitate analysis inthese methods, the activatable antibodies can be detectably labeled, andcan be bound to a support (e.g., a solid support, such as a slide orbead). The detectable label can be positioned on a portion of theactivatable antibody that is not released following cleavage, forexample, the detectable label can be a quenched fluorescent label orother label that is not detectable until cleavage has occurred. Theassay can be conducted by, for example, contacting the immobilized,detectably labeled activatable antibodies with a sample suspected ofcontaining an enzyme and/or reducing agent for a time sufficient forcleavage to occur, then washing to remove excess sample andcontaminants. The presence or absence of the cleaving agent (e.g.,enzyme or reducing agent) in the sample is then assessed by a change indetectable signal of the activatable antibodies prior to contacting withthe sample e.g., the presence of and/or an increase in detectable signaldue to cleavage of the activatable antibody by the cleaving agent in thesample.

Such detection methods can be adapted to also provide for detection ofthe presence or absence of a target that is capable of binding the AB ofthe activatable antibodies when cleaved. Thus, the assays can be adaptedto assess the presence or absence of a cleaving agent and the presenceor absence of a target of interest. The presence or absence of thecleaving agent can be detected by the presence of and/or an increase indetectable label of the activatable antibodies as described above, andthe presence or absence of the target can be detected by detection of atarget-AB complex e.g., by use of a detectably labeled anti-targetantibody.

Activatable antibodies are also useful in in situ imaging for thevalidation of activatable antibody activation, e.g., by proteasecleavage, and binding to a particular target. In situ imaging is atechnique that enables localization of proteolytic activity and targetin biological samples such as cell cultures or tissue sections. Usingthis technique, it is possible to confirm both binding to a given targetand proteolytic activity based on the presence of a detectable label(e.g., a fluorescent label).

These techniques are useful with any frozen cells or tissue derived froma disease site (e.g. tumor tissue) or healthy tissues. These techniquesare also useful with fresh cell or tissue samples.

In these techniques, an activatable antibody is labeled with adetectable label. The detectable label may be a fluorescent dye, (e.g.Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), anear infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, ahapten, a radioactive marker, biotin and an amplification reagent suchas streptavidin, or an enzyme (e.g. horseradish peroxidase or alkalinephosphatase).

Detection of the label in a sample that has been incubated with thelabeled, activatable antibody indicates that the sample contains thetarget and contains a protease that is specific for the CM of theactivatable antibody. In some embodiments, the presence of the proteasecan be confirmed using broad spectrum protease inhibitors such as thosedescribed herein, and/or by using an agent that is specific for theprotease, for example, an antibody such as A11, which is specific forthe protease matriptase (MT-SP1) and inhibits the proteolytic activityof matriptase; see e.g., International Publication Number WO2010/129609, published 11 Nov. 2010. The same approach of using broadspectrum protease inhibitors such as those described herein, and/or byusing a more selective inhibitory agent can be used to identify aprotease or class of proteases specific for the CM of the activatableantibody. In some embodiments, the presence of the target can beconfirmed using an agent that is specific for the target, e.g., anotherantibody, or the detectable label can be competed with unlabeled target.In some embodiments, unlabeled activatable antibody could be used, withdetection by a labeled secondary antibody or more complex detectionsystem.

Similar techniques are also useful for in vivo imaging where detectionof the fluorescent signal in a subject, e.g., a mammal, including ahuman, indicates that the disease site contains the target and containsa protease that is specific for the CM of the activatable antibody.

These techniques are also useful in kits and/or as reagents for thedetection, identification or characterization of protease activity in avariety of cells, tissues, and organisms based on the protease-specificCM in the activatable antibody.

In some embodiments, in situ imaging and/or in vivo imaging are usefulin methods to identify which patients to treat. For example, in in situimaging, the activatable antibodies are used to screen patient samplesto identify those patients having the appropriate protease(s) andtarget(s) at the appropriate location, e.g., at a tumor site.

In some embodiments, in situ imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableantibody of the disclosure. For example, patients that test positive forboth the target and a protease that cleaves the substrate in thecleavable moiety (CM) of the activatable antibody being tested (e.g.,accumulate activated antibodies at the disease site) are identified assuitable candidates for treatment with such an activatable antibodycomprising such a CM. Likewise, patients that test negative for eitheror both of the target and the protease that cleaves the substrate in theCM in the activatable antibody being tested using these methods areidentified as suitable candidates for another form of therapy (i.e., notsuitable for treatment with the activatable antibody being tested). Insome embodiments, such patients that test negative with respect to afirst activatable antibody can be tested with other activatableantibodies comprising different CMs until a suitable activatableantibody for treatment is identified (e.g., an activatable antibodycomprising a CM that is cleaved by the patient at the site of disease).

In some embodiments, in vivo imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableantibody of the disclosure. For example, patients that test positive forboth the target and a protease that cleaves the substrate in thecleavable moiety (CM) of the activatable antibody being tested (e.g.,accumulate activated antibodies at the disease site) are identified assuitable candidates for treatment with such an activatable antibodycomprising such a CM. Likewise, patients that test negative areidentified as suitable candidates for another form of therapy (i.e., notsuitable for treatment with the activatable antibody being tested). Insome embodiments, such patients that test negative with respect to afirst activatable antibody can be tested with other activatableantibodies comprising different CMs until a suitable activatableantibody for treatment is identified (e.g., an activatable antibodycomprising a CM that is cleaved by the patient at the site of disease).

Pharmaceutical Compositions

The conjugated antibodies, activatable antibodies and/or conjugatedactivatable antibodies of the disclosure (also referred to herein as“active compounds”), and derivatives, fragments, analogs and homologsthereof, can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the conjugatedantibody, activatable antibody and/or conjugated activatable antibodyand a pharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Suitable examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In someembodiments, it will be desirable to include isotonic agents, forexample, sugars, polyalcohols such as manitol, sorbitol, sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent thatdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Ability of Synovial Fluid to Activate Quenched ProbesComprising Substrates of the Disclosure

This Example demonstrates the ability of synovial fluid samples tocleave MMP substrate sequences of the disclosure. In particular, the MMPcleavable sequences were tested in the context of an activatableantibody construct comprising a masking moiety linked to an anti-IL-6Rantibody sequence via a linker region that includes the MMP cleavablesequence being evaluated.

The following MMP-cleavable activatable antibodies were incubated withsynovial fluid:

4792¹⁰⁴¹⁹AV1 amino acid (SEQ ID NO: 115)QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSGISSGLSSGGSDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC4792¹⁰⁴¹⁹AV1 nucleotide (SEQ ID NO: 116)Caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcggctcgagcggtggcagcggtggctctggtggctcaggtattagtagtggtcttagcagtggcggttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcaccatcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaaaagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcagtggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgccacatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgt4792⁵⁵⁹AV1 amino acid (SEQ ID NO: 117)QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSQNQALRMAGGSDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC4792⁵⁵⁹AV1 nucleotide (SEQ ID NO: 118)caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcggctcgagcggtggcagcggtggctctggtggctcacagaatcaggcattacgtatggcaggcggttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcaccatcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaaaagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcagtggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgccacatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgt4792⁶⁰¹AV1 amino acid (SEQ ID NO: 119)QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSAQNLLGMVGGSDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC4792⁶⁰¹AV1 nucleotide (SEQ ID NO: 120)Caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcggctcgagcggtggcagcggtggctctggtggctcagcacagaatctgttaggtatggtaggcggttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcaccatcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaaaagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcagtggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgccacatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgt4792³⁴⁵⁷AV1 amino acid (SEQ ID NO: 121)QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSSTFPFGMFGGSDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC4792³⁴⁵⁷AV1 nucleotide (SEQ ID NO: 122)Caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcggctcgagcggtggcagcggtggctctggtggctcaagtacatttccattcggtatgttcggcggttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcaccatcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaaaagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcagtggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgccacatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgt4792³⁴⁵⁸AV1 amino acid (SEQ ID NO: 123)QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSPVGYTSSLGGSDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC4792³⁴⁵⁸AV1 nucleotide (SEQ ID NO: 124)Caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcggctcgagcggtggcagcggtggctctggtggctcacctgttggatatacgagtagtctgggcggttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcaccatcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaaaagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcagtggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgccacatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgt4792³⁴⁶³AV1 amino acid (SEQ ID NO: 125)QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSDWLYWPGIGGSDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC4792³⁴⁶³AV1 nucleotide (SEQ ID NO: 66)Caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcggctcgagcggtggcagcggtggctctggtggctcagactggttatactggcctggtattggcggttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcaccatcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaaaagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcagtggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgccacatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgt

The extent of activatable antibody activation was determined by an ELISAformat that measured the ability of the activatable antibody, followingincubation in synovial fluid, to bind to human IL6R as compared to thebinding of anti-IL6R parental antibody to IL6R. Briefly, Nunc Maxisorpplates were coated overnight at 4° C. with 100 μl/well(microliters/well) of a 500-ng/mL solution of human IL6R (R and DSystems, Cat No. 227-SR/CF) in PBS, pH 7.4. Plates were washed 3 timeswith PBST (PBS, pH 7.4, 0.05% Tween-20). Wells were then blocked with200 μl/well, 2% NFDM (non-fat dry milk) in PBST for 2 hours at roomtemperature. The IL6R-coated plates were washed 3 times with PBST (PBS,pH 7.4, 0.05% Tween-20). A dilution series of each activatableantibody—synovial fluid reaction mixture, as well as a dilution seriesof the parental anti-IL6R antibody, was added to appropriate wells ofthe IL6R-coated ELISA plate. The plates were incubated 1 hour at roomtemperature, and then washed 3 times with PBST (PBS, pH 7.4, 0.05%Tween-20). One hundred μl/well 1:3000 dilution goat-anti-human IgG (Fabspecific, Sigma Cat No. A0293) in 2% NFDM-PBST was added, and the plateincubated for 1 hour at room temperature. The plates were washed 6 timeswith PBST (PBS, pH 7.4, 0.05% Tween-20) and then developed with TMB and1N HCl.

Table 6 provides the results of this experiment. The data indicate thatanti-IL6R activatable antibodies comprising the substrates in Table 6are cleaved by at least some synovial fluid samples (SyF) obtained fromRA patients.

TABLE 6 Activatable Antibody Activation Substrate/ Activation ActivationIncidence Sequence in vivo in SyF in SyF 10419 <5% >30% 3/3 ISSGLSS (SEQID NO: 159) 559 <5% 20% 3/3 QNQALRMA (SEQ ID NO: 15) 601 <5% >30% 3/3AQNLLGMV (SEQ ID NO: 16) 3457 10% >50% 3/3 STFPFGMF (SEQ ID NO: 17) 345810% 20% 3/3 PVGYTSSL (SEQ ID NO: 18) 3463 <5% >30% 3/3 DWLYWPGI (SEQ IDNO: 19)

Example 2 Activatable Anti-EGFR Antibody with MMP-Cleavable Substrate toInhibit Tumor Growth

This Example demonstrates the ability of an activatable anti-EGFRantibody that contains a masking moiety comprising the amino acidsequence CISPRGCPDGPYVMY (SEQ ID NO: 160), a cleavage moiety comprisingthe MMP14 substrate 520 (also referred to herein as MN520) ISSGLLSS (SEQID NO: 14), and the heavy chain (SEQ ID NO: 56) and light chain (SEQ IDNO: 59) of the anti-EGFR antibody C225v5, where the entire activatableantibody construct is referred to herein as Pb-MN520, to inhibit tumorgrowth in the H292 xenograft lung cancer model. The configuration of thelight chain of the activatable antibody was masking moiety—MMPsubstrate—light chain of C225v5.

FIG. 1A is a graph depicting the effects seen in H292 xenografttumor-bearing mice that were treated using Pb-520 (12.5 mg/kg, solidblue line) and IVIG (12.5 mg/kg, green dashed line) dosed at differenttimes. Data are presented as mean tumor volume±SEM. FIG. 1B is a graphdepicting systemic stability of the Pb-520 activatable antibody in H292tumor bearing mice. Blood samples were taken through retro-orbitalbleeds at Day 7 and the circulating stability of substrate 520 wasdetermined by analysis of IgG pull-downs with capillary electrophoresis(GXII; Caliper LifeSciences). Concentrations of cleaved and uncleavedlight chain were determined using LabChip GX software (CaliperLifeSciences).

Example 3 Materials and Methods

Reagents and Strains:

Streptavidin-conjugated phycoerythrin (SA-PE) (Invitrogen, LifeTechnologies) was used without modifications. Human MMP9 (Research &Diagnostics Systems, Inc.) was activated following the supplied protocoland used without modifications. Human MMP14 (Research & DiagnosticsSystems, Inc.) was activated following the supplied protocol and usedwithout modifications. Human Plasmin (Haematologic Technologies Inc.)was used without modifications. Human tPA (Molecular Innovations) wasused without modifications. YPet fused to the SH3 domain of Mona(monocytic adaptor protein) was produced at CytomX Therapeutics and usedwithout modifications. MMP14 Buffer HCM (50 mM HEPES (pH 6.8), 10 mMCaCl₂, 0.5 mM MgCl₂), was used. MMP9 Buffer TCNB (50 mM Tris-HCl, 10 mMCaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5) was used. PlasminBuffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 0.01% Tween20 and 1 mM EDTAwas used. TBST (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween20, pH 7.4) wasused. E. coli MC1061 (Casadaban et al., JMB 138(2):179-207 (1980) wasused. All bacterial growth was performed at 37° C. with vigorous shakingin Luria-Bertani broth (LB) supplemented with 34 μg/mL chloramphenicol,unless another antibiotic is specified.

Substrate Cleavage and Scaffold Stability Analysis:

For screening and clone analysis, overnight cultures were subcultured bydilution into fresh medium (1:50) and grown for 1.5-2 hours. Thesubculture was then induced with 0.04% arabinose and incubated withshaking at 37° C. for 45 minutes to 1 hour. To stop further growth cellswere incubated on ice for 15 minutes to 1 hour. Cell aliquots wereharvested and washed with PBS (pH 7.4). Cells were pelleted bycentrifugation, the supernatant removed and the cells resuspended inreaction buffer containing the enzyme; the reaction mixture wasincubated at 37° C. static. To stop the reaction, cells were removed anddiluted 10-fold in PBS, pelleted by centrifugation, and resuspended inPBS containing either (CLiPS) SA-PE (20 μg/mL) or YPet-MONA (50 nM).After incubation on ice (30 min), cells were washed with PBS andanalyzed using a FACSAria™ cell sorter.

For MMP9 protease cleavage assays, cultures were induced for 45 minutesto 1 hour. The reaction buffer for MMP9 was TCNB. Assays for MMP9hydrolysis were performed after fresh cells were incubated with 5 nM-25nM MMP9 for 1 hr. Background hydrolysis of the regions flanking thesubstrate site (using platform eCLiPS3.0-NSUB_SP described in PCT patentapplication PCT/US13/54378, filed Aug. 9, 2013, which was published asInternational Publication No. WO 2014/026136 on 13 Feb. 2014, thecontents of which are hereby incorporated by reference in theirentirety) was measured under each reaction condition to ensure thathydrolysis occurred in the designated substrate region.

For MMP14 protease cleavage assays, cultures were induced for 45 minutesto 1 hour. The reaction buffer for MMP14 was HCM. Assays for MMP14hydrolysis were performed after reactions with 3 nM-250 nM MMP14 or 1hr. Background hydrolysis of the regions flanking the substrate site(using platform eCLiPS3.0-NSUB_SP described herein) was measured undereach reaction condition to ensure that hydrolysis occurred in thedesignated substrate region.

For human plasmin stability assays, platform eCLiPS3.0-NSUB_SP is used;cultures are induced for 45 minutes to 1 hr. The reaction buffer forplasmin is 50 mM Tris-HCl pH 7.5 supplemented with 100 mM NaCl, 0.01%Tween20 and 1 mM EDTA. Assays for plasmin hydrolysis are performed afterreactions with plasmin for 1 hr.

For human tPA stability assays, platform eCLiPS3.0-NSUB_SP is used;cultures are induced for 45 minutes to 1 hr. The reaction buffer for tPAis TBST. Assays for tPA hydrolysis are performed after reactions withtPA for 1 hr.

Amino and Carboxy Terminus Labeling Conditions:

Streptavidin conjugated phycoerythrin (SAPE) was used for labelingstreptavidin binding affinity ligand on the N-termini of CPX.Fluorescent protein YPet fused to the SH3 domain of Mona was used forlabeling the MONA binding affinity ligand on the C-termini of CPX. Foroptimum labeling of cells without protease reaction, the cells wereincubated for 30 min at 4° C. with SAPE (20 μg/mL) or YPet-MONA (50 nM).For the described example below 30 min incubation was used.

Kinetic Data Analysis:

The extent of conversion of cell surface displayed peptide substrateswas measured directly, using flow cytometry to measure changes in meanfluorescence of clonal cell populations upon protease treatment.Specifically, for each sample, conversion was determined by flowcytometry analyses using the relationship

$\begin{matrix}{{{Conversion} = \frac{{F\; L_{-}} - {F\; L_{+}}}{{F\; L_{-}} - {F\; L_{0}}}}\;} & \lbrack 1\rbrack\end{matrix}$

where (FL⁻) is the fluorescence after incubating without enzyme, (FL₊)is fluorescence after incubation with enzyme, and (FL₀) is fluorescenceof unlabeled cells. Given that the expected substrate concentrationsthat were used are significantly below the expected K_(M) of thesubstrate for the target protease, the Michaelis-Menten model simplifiesto:

$\begin{matrix}{\frac{\lbrack S\rbrack}{t} \approx {{\frac{k_{cat}}{k_{M}}\lbrack S\rbrack}\lbrack E\rbrack}} & \lbrack 2\rbrack\end{matrix}$

allowing substrate conversion to be expressed as

$\begin{matrix}{{Conversion} = {1 - {\exp( {{- \frac{k_{cat}}{k_{M}}} \cdot \lbrack E\rbrack \cdot t} )}}} & \lbrack 3\rbrack\end{matrix}$

where [S] is the substrate concentration, [E] is enzyme concentrationand t is time. To determine the second order rate constant(k_(cat)/K_(M)), equation [3] was simplified to:

$\frac{k_{cat}}{K_{m}} = {{- {\ln ( {1 - C} )}}/( {t*p} )}$

where C is product conversion, t is time and p is proteaseconcentration.

Sequence Data Analysis—Directed Families:

Substrates were submitted to Ion Torrent™ sequencing (see, e.g.,Rothenberg, J M, Nature 475, 348-352). Raw Ion Torrent reads werecropped by invariant vector sequences to obtain just the variablepeptide insert. Insert sequences were translated, and sequences withstop codons were excluded from further analysis. The frequency of eachsequence was obtained by number of times observed out of all viablepeptide reads observed. Enrichment of sequences was obtained bycomparison of observed frequency of each sequence post selection to thefrequency of each sequence pre-selection. Individual sequences wereidentified and isolated from these data, and sequences were aligned inCLC main lab (CLC Main Workbench 6.6.2, available online). The alignmentfile was imported to Jalview (see, e.g., Waterhouse, A. M., et al.,2009, Bioinformatics 9, 1189-1191), and an average distance tree wasassembled using the BLOSUM62 algorithm (S Henikoff S et al., 1992, ProcNatl Acad Sci USA. 89, 10915-10919). The restricted group of sequencesincludes members of the cluster closest to the sequence of interest. Theextended group of sequences includes the restricted group of sequencesplus members of the branch that shares the closest common ancestor(where applicable).

Example 4 Selection and Characterization of Substrate Pools in aPlatform Scaffold

The use of multi-copy substrate display on whole cells enabled selectionof populations of substrates cleaved by MMP9. Selections were performedas described in U.S. Pat. No. 7,666,817 B2, issued Feb. 23, 2010, usingrecombinant human MMP9. Background hydrolysis of the regions flankingthe substrate site (using platform eCLiPS3.0-NSUB_SP) was measured undereach reaction condition to ensure that hydrolysis occurred in thedesignated substrate region. Selected pools were tested with MMP9 andMMP14. FIGS. 2A and 2B show cleavage of pool SMP87 by MMP9 at 5 nM inTCNB buffer.

Example 5 Characterization of Substrate Cleavage Kinetics in thePlatform Scaffold

The use of multi-copy substrate display on whole cells enabled simpleand direct quantitative characterization of cleavage kinetics.Consequently, flow cytometry was used to rank individual isolated cloneson the basis of substrate conversion, and clones were identified by DNAsequencing. In this way, the extent of conversion for each clone couldbe determined at several different protease concentrations and fit to aMichaelis-Menten model (Kinetic Data Analysis Section). The observedsecond order rate constant (k_(cat)/K_(M)) was determined for eachsubstrate versus MMP9. Background hydrolysis of the regions flanking thesubstrate site (using platform eCLiPS3.0-NSUB_SP), was measured undereach reaction condition to ensure that hydrolysis occurred in thedesignated substrate region. For example, FIGS. 3A and 3B show cleavageof a substrate comprising amino acid sequence VAGRSMRP (SEQ ID NO: 484)by 5 nM MMP9 in TBST.

Example 6 Correlation of Next Generation Sequencing Frequency andSubstrate Cleavage Kinetics in the Platform Scaffold

Final pools of enriched substrates were sequenced using Ion TorrentNext-Generation Sequencing. Raw Ion Torrent reads were cropped byinvariant vector sequences to obtain just the variable peptide insert.Insert sequences were translated, and sequences with stop codons wereexcluded from further analysis. A selection of clones (displaying arange of frequencies) was selected for functional analysis. Selectedclones were cleaved with human MMP9, and a k_(cat)/K_(M) was determinedfor each. The log of the clone copy number in the pool was then plottedversus the log of the k_(cat)/K_(M). FIG. 4 shows the correlationbetween frequency of particular cleavage moieties (Copy Number) andtheir abilities to be cleaved by MMP9 (MMP9 k_(cat)/K_(M) M⁻¹ s⁻¹).

Example 7 Selection and Characterization of Substrate Pools in aPlatform Scaffold

The use of multi-copy substrate display on whole cells enabled selectionof populations of substrates cleaved by MMP14. Selections were performedas described in U.S. Pat. No. 7,666,817 B2, using recombinant humanMMP14. Background hydrolysis of the regions flanking the substrate site(using platform eCLiPS3.0-NSUB_SP) was measured under each reactioncondition to ensure that hydrolysis occurred in the designated substrateregion. Selected pools were tested with MMP9 and MMP14. FIGS. 5A and 5Bshow cleavage of pool SMP39 by MMP14 at 60 nM in HCM buffer.

Example 8 Characterization of Substrate Cleavage Kinetics in thePlatform Scaffold

The use of multi-copy substrate display on whole cells enabled simpleand direct quantitative characterization of cleavage kinetics.Consequently, flow cytometry was used to rank individual isolated cloneson the basis of substrate conversion, and clones were identified by DNAsequencing. In this way, the extent of conversion for each clone couldbe determined at several different protease concentrations and fit to aMichaelis-Menten model (Kinetic Data Analysis Section). The observedsecond order rate constant (k_(cat)/K_(M)) was determined for eachsubstrate versus MMP14. Background hydrolysis of the regions flankingthe substrate site (using platform eCLiPS3.0-NSUB_SP), was measuredunder each reaction condition to ensure that hydrolysis occurred in thedesignated substrate region. For example, FIGS. 6A and 6B show cleavageof a substrate comprising amino acid sequence QNQALRMA (SEQ ID NO: 15)by 30 nM MMP14 in HCM buffer.

Example 9 In Vitro Substrate Activity in Activatable Antibodies

This Example demonstrates the in vitro activity of substrates of thedisclosure when they are incorporated into activatable antibodies.

Several substrates identified in these studies were inserted intoactivatable antibodies having the 3954 mask and C225v5 variant ofcetuximab, which is described in PCT Publication No. WO 2013/163631, andwhich is incorporated herein by reference in its entirety.

The ability of substrates in the resultant activatable antibodies to becleaved by MMP9 or MMP14 was determined as follows. MMP9 proteasedigests were performed in TCNB, 50 mM Tris-HCl, 10 mM CaCl₂, 150 mMNaCl, 0.05% (w/v) Brij-35, pH 7.5. MMP14 digests were performed in 50 mMHEPES (pH 6.8), 10 mM CaCl₂, 0.5 mM MgCl₂. Varying concentrations ofactive site titrated MMP9 or MMP14 were combined with a fixedactivatable antibody concentration to maintain a substrate to proteaseratio of at least 50. Samples comprising MMP9 substrates were incubatedat 37° C. for up to 24 hr. Samples comprising MMP14 substrates wereincubated at 37° C. for 4 hr. To stop the reaction, 5 μl of the digestwas added to 7 μl of HT Protein Express Sample Buffer (CaliperLifeSciences) containing 20 mM 2-Mercaptoethanol for 10 minutes at 95°C. After heat denaturation, 32 μl of ddH₂O was added and samplesanalyzed on a LabChip GXII per manufacturer's instructions. The LabChipGXII software was used to quantify light chain peak area. Productconversion was calculated by plugging the light chain peak areas intothe following equation: cleaved LC/(cleaved LC+uncleaved LC), LC=lightchain. k_(cat)/K_(M) values were determined with the following equation

$\frac{k_{cat}}{K_{m}} = {{- {\ln ( {1 - C} )}}/( {t*p} )}$

where C is product conversion, t is time (s), and p is proteaseconcentration (M), which assumes that the substrate concentration isbelow the K_(M) and in excess of the protease concentration.

Resultant activatable antibodies comprising substrates selected forcleavage by MMP14 tested for cleavage by MMP14 had k_(cat)/K_(M) valuesranging from about 400 to 60,000 M⁻¹ s⁻¹ for MMP14. Resultantactivatable antibodies comprising substrates selected for cleavage byMMP9 tested for cleavage by MMP9 were cleaved by MMP9.

Example 10 Substrate Stability of Activatable Antibodies In Vivo

This Example demonstrates the in vivo stability of substrates of thedisclosure when they are incorporated into activatable antibodies andinjected into mice.

Three nude mice (Crl:NU-Foxn1nu) received a single IP dose of eachactivatable antibody at 12.5 mg/kg on Day 0. Mice were euthanized on day4 (˜96 h post-dose) by CO₂ asphyxiation, and blood was collectedimmediately as plasma-EDTA and stored at −80° C.

Activatable antibodies were purified from plasma by anti-human IgGimmunoprecipitation using magnetic beads. Eluted activatable antibodieswere prepared for analysis by capillary electrophoresis as described inthe k_(cat)/K_(M) section. Briefly, 5 μl of eluted IgG was added to 7 μlProtein Express Sample Buffer with 2-mercaptoethanol. Quantification ofcirculating stability was identical to quantification of productconversion.

Of ten activatable antibodies comprising substrates of the disclosureselected for cleavage by MMP14, seven demonstrated less than 20%cleavage in the collected plasma samples. Of seven activatableantibodies comprising substrates of the disclosure selected for cleavageby MMP9, four demonstrated no more than 20% cleavage in the collectedplasma samples.

Example 11 Materials and Methods

Reagents and Strains:

Human MMP9 (catalog no. 911-MP, Research & Diagnostics Systems, Inc.)was activated following the supplied protocol and used withoutmodifications. Human MMP14 (catalog no. 918-M), Research & DiagnosticsSystems, Inc.) was activated following the supplied protocol and usedwithout modifications. Human Plasmin (catalog no. HCPM-0140,Haematologic Technologies Inc.) was used without modifications. Anti-EEmonoclonal antibody (Covance, Princeton, N.J.) was labeled with Alexa647 (Life Sciences) and used with no other modifications (named EE647).E. coli MC1061 or MC1061 derived strains (DH10β) were used for allexperiments (Casadaban et al., JMB 138(2):179-207 (1980)). All bacterialgrowth was performed at 37° C. with vigorous shaking in Luria-Bertanibroth (LB) supplemented with 34 μg/mL chloramphenicol (cm), unlessanother antibiotic is specified.

Display Platforms:

Display platforms, each engineered to contain an 8-amino acid substrateof the embodiments, were produced and used as described in InternationalPublication No. WO 2014/026136, published 13 Feb. 2014, the contents ofwhich are hereby incorporated by reference in their entirety. The aminoacid sequence of the mature (i.e., without a signal peptide)CYTX-DP-XXXXXXXX display platform (SEQ ID NO: 512) is shown in FIG. 7A.XXXXXXXX indicates the location into which each substrate is inserted.The amino acid sequence of CYTX-DP-XXXXXXXX display platform alsoincluding its signal peptide, i.e., SP-CYTX-DP-XXXXXXXX display platform(SEQ ID NO: 513) is shown in FIG. 7B.

CYTX-DP-XXXXXXXX Display Platform:

(SEQ ID NO: 512) GQSGQEYMPMEGGSGQXXXXXXXXSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGHHHHHHHH

SP-CYTX-DP-XXXXXXXX Display Platform:

(SEQ ID NO: 513) MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQXXXXXXXXSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSG GSSGQAAAGHHHHHHHH

Substrate Cleavage and Cleavage Kinetics Analysis:

For clone analysis, overnight cultures were subcultured by dilution intofresh medium (1:40) and grown for 1.5-2 hours. The subculture was theninduced with 0.04% arabinose and incubated with shaking at 37° C. for 40minutes to 1 hour. To stop further growth, cells were then incubated onice for 15 minutes to 1 hour. Cell aliquots were harvested and washedwith PBS (pH 7.4). Cells were pelleted by centrifugation, thesupernatant removed and the cells resuspended in reaction buffercontaining the enzyme; the reaction mixture was incubated at 37° C. withshaking To stop the reaction cells were removed and diluted 10-fold inPBS, pelleted by centrifugation, and resuspended in PBS containing EE647(20 micrograms per ml (also referred to herein as ug/ml or μg/ml)).After incubation on ice (1 hour), cells were washed with PBS andanalyzed using an Accuri C6 cell sorter.

For MMP9 protease cleavage assays, cultures were induced for 45 minutes.The reaction buffer for MMP9 was 50 mM Tris-HCl, pH 7.4, supplementedwith 150 mM NaCl, 10 mM CaCl₂ and 0.05% (w/v) Brij-35. Assays for MMP9hydrolysis, were performed after cleavage with 5 nM-150 nM MMP9 for 1hour. Background hydrolysis of the regions flanking the substrate site(using, e.g., CYTX-DP-NSUB, a display platform in which the “Substrate”is non-cleavable linker GGGSGGGS) was measured under each reactioncondition to ensure that hydrolysis occurred in the designated substrateregion.

For MMP14 protease cleavage assays, cultures were induced for 45minutes. The reaction buffer for MMP14 was 50 mM HEPES, pH 6.8,supplemented with 10 mM CaCl₂ and 0.5 mM MgCl₂. Assays for MMP14hydrolysis, were performed after cleavage with 5 nM-150 nM MMP14 for 1hr. Background hydrolysis of the regions flanking the substrate site(using, e.g., CYTX-DP-NSUB) was measured under each reaction conditionto ensure that hydrolysis occurred in the designated substrate region.

For human plasmin stability assays, cultures were induced for 45minutes. The reaction buffer for plasmin was 50 mM Tris-HCl pH 7.4,supplemented with 100 mM NaCl, 0.01% Tween20 and 1 mM EDTA. Assays forplasmin hydrolysis were performed after cleavage with 500 pM plasmin for1 hr. Background hydrolysis of the regions flanking the substrate site(using, e.g., CYTX-DP-NSUB) was measured under each reaction conditionto ensure that hydrolysis occurred in the designated substrate region.

Amino and Carboxyl Terminus Labeling Conditions:

Alexa-647 conjugated anti-EE antibody (EE647) was used for labeling EEbinding affinity ligand on the N-termini of the CYTX-DP displayplatform. Alexa-647 conjugated anti-His antibody (His647) was used forlabeling the 8His binding affinity ligand on the C-termini of theCYTX-DP display platform. For optimum labeling of cells without proteasereaction, the cells were incubated for 1 hour at 4° C. with EE647 (20μg/mL) or His647 (2 μg/mL). For the example described below, a 1-hourincubation was used.

Kinetic Data Analysis:

The extent of conversion of cell surface displayed peptide substrateswas measured directly, using flow cytometry to measure changes in meanfluorescence of clonal cell populations upon protease treatment.Specifically, for each sample, conversion was determined by flowcytometry analyses using the relationship

$\begin{matrix}{{Conversion}_{CLiPS} = \frac{{F\; L_{-}} - {F\; L_{+}}}{{F\; L_{-}} - {F\; L_{0}}}} & \lbrack 1\rbrack\end{matrix}$

where (FL⁻) is the fluorescence after incubating without enzyme, (FL₊)is fluorescence after incubation with enzyme, and (FL₀) is fluorescenceof unlabeled cells. Given that the expected substrate concentrationsthat were used are significantly below the expected K_(M) of thesubstrate for the target protease, the Michaelis-Menten model simplifiesto

$\begin{matrix}{\frac{\lbrack S\rbrack}{t} \approx {{\frac{k_{cat}}{k_{M}}\lbrack S\rbrack}\lbrack E\rbrack}} & \lbrack 2\rbrack\end{matrix}$

allowing substrate conversion to be expressed as

$\begin{matrix}{{Conversion}_{MM} = {1 - {\exp( {{- \frac{k_{cat}}{k_{M}}} \cdot \lbrack E\rbrack \cdot t} )}}} & \lbrack 3\rbrack\end{matrix}$

where [S] is the substrate concentration, [E] is enzyme concentrationand t is time. To determine the second order rate constant(k_(cat)/K_(M)), the time dependent conversion for each substrate wasfit to equation [3].

Example 12 Characterization of Substrate Cleavability in CYTX-DP DisplayPlatform

This Example demonstrates the ability of substrates of the embodimentsto be cleaved by MMP but not by plasmin.

The use of multi-copy substrate display on whole cells enabled simpleand direct quantitative characterization of cleavage kinetics. Clonesencoding substrates were identified by DNA sequencing and subcloned intothe CYTX-DP display platform such that the expressed display platformcontained the 8-amino acid substrate in place of XXXXXXXX. Individualsubstrate displaying clones (127 independent substrate-containingdisplay platforms in total) were assessed for cleavage by either MMP9 orMMP14 (target proteases, i.e., the proteases used to select thesubstrate) and plasmin (off-target protease); turnover was determined byflow cytometry. Thirty-one of the MMP9-selected substrates were selectedfrom the same pool that was the source of substrates comprising aminoacid sequences SEQ ID NOs: 17, 18, 19, 20, 21, 22, or 23 (MMP9substrates from pool). Nine of the MMP9-selected substrates compriseconsensus amino acid sequences SEQ ID NOs: 328, 336, 337, 338, 339, 348,349, 350 or 351 (MMP9 consensus sequences). Thirty-eight of theMMP14-selected substrates were selected from the same pool that was thesource of substrates comprising amino acid sequences SEQ ID NOs: 14, 15,16, 24, 25, 26, 27, 28, 29, 30, or 33 (MMP14 substrates from 1^(st)pool). Ten of the MMP14-selected substrates were selected from the samepool that was the source of substrates comprising amino acid sequencesSEQ ID NOs: 31 or 32 (MMP14 substrates from 2^(nd) pool). Thirty-nine ofthe MMP14-selected substrates were chosen from consensus amino acidsequences SEQ ID NOs: 364-370, 379-393, 402-409, 420-424, 434-435,450-452, 457, 470-472, 474, or 483 (MMP14 consensus sequences).

In this way, the extent of cleavage for each clone could be determinedand the data aggregated to determine a percent of clones that arecleaved by the target protease and not the off-target protease.Background hydrolysis of the regions flanking the substrate site (using,e.g., CYTX-DP-NSUB) was measured under each reaction condition to ensurethat hydrolysis occurred in the designated substrate region. Results arepresented in Table 9.

TABLE 9 Summary statistics of substrate cleavability >20% Cleavage with<20% Cleavage Discovery 50 nM MMP9 or with 500 pM effort Substrate GroupMMP14 Plasmin MMP9- All MMP9 35% (14 of 40)  85% (34 of 40) selectedsubstrates tested Substrates MMP9 substrates 39% (12 of 31)  84% (26 of31) from pool MMP9 consensus 22% (2 of 9)  89% (8 of 9) substratesMMP14- All MMP14 85% (74 of 87)  94% (82 of 87) selected substratestested Substrates MMP14 79% (38 of 48)  94% (45 of 48) substrates from1^(st) and 2^(nd) pools MMP14 79% (30 of 38)  95% (36 of 38) substratesfrom 1^(st) pool MMP14 80% (8 of 10) 100% (9 of 10) substrates from2^(nd) pool MMP14 92% (36 of 39)  95% (37 of 39) consensus substratesCombined Total 69% (88 of 127)  91% (116 of 127) MMP9 and MMP14

Table 9 depicts (a) the percentage of MMP9-selected substrates tested inthe CYTX-DP display platform that exhibited at least 20% cleavage whenincubated with 50 nM human MMP9 (catalog no. 911-MP, Research &Diagnostics Systems, Inc., activated following the supplied protocol andused without modifications) for 1 hour at 37° C. in 50 mM Tris-HCl, pH7.4, supplemented with 150 mM NaCl, 10 mM CaCl₂, and 0.05% (w/v) Brij-35(>20% Cleavage with 50 nM MMP9); (b) the percentage of MMP14-selectedsubstrates tested in the CYTX-DP display platform that exhibited atleast 20% cleavage when incubated with 50 nM human MMP14 (catalog no.918-MP, Research & Diagnostics Systems, Inc., activated following thesupplied protocol and used without modifications) for 1 hour at 37° C.in 50 mM HEPES, pH 6.8, supplemented with 10 mM CaCl₂, and 0.5 mM MgCl₂(>20% Cleavage with 50 nM MMP14); and (c) the percentage ofMMP9-selected or MMP-14-selected substrates tested in the CYTX-DPdisplay platform that exhibited less than 20% cleavage when incubatedwith 500 pM human plasmin (catalog number HCPM-0140, HaematologicTechnologies, Inc., used without modifications) for 1 hour at 37° C. in50 mM Tris-HCl, pH 7.4, supplemented with 100 mM NaCl, 0.01% Tween20 and1 mM EDTA (<20% cleavage with 500 pM plasmin).

Example 13 Characterization of Substrate Cleavage Kinetics in CYTX-DPDisplay Platforms

This Example demonstrates the cleavage kinetics of various substrates.

The use of multi-copy substrate display on whole cells enabled simpleand direct quantitative characterization of cleavage kinetics. Cloneswere identified by DNA sequencing and subcloned into theCYTX-DP-XXXXXXXX display platform as described in the preceding example.Seventy-two individual substrate-displaying clones were assessed forcleavage and a subset were chosen to assess cleavage kinetics by theirtarget protease. The extent of conversion for each clone could bedetermined at several different protease concentrations and fit to theMichaelis-Menten model described herein. Observed k_(cat)/K_(M) valueswere then plotted versus frequency of the clone within the substratepool and a correlation between frequency and k_(cat)/K_(M) was seen.Background hydrolysis of the regions flanking the substrate site (using,e.g., CYTX-DP-NSUB) was measured under each reaction condition to ensurethat hydrolysis occurred in the designated substrate region. Results arepresented in Table 10.

TABLE 10 Summary statistics of substrate kinetics Target Target ProteaseProtease Substrate Target Protease k_(cat)/K_(M) > k_(cal)/K_(M) > Groupk_(cat)/K_(M) > 1 × 10E2 1 × 10E3 1 × 10E4 MMP9 All MMP9 100% (16 of 16)100% (16 of 16) 63% (10 of 16) Substrates substrates tested MMP9 100%(15 of 15) 100% (15 of 15) 67% (10 of 15) substrates from pool MMP9 100%(1 of 1) 100% (1 of 1)  0% (0 of 1) consensus substrates MMP14 AllMMP-14 100% (55 of 55)  98% (54 of 55) 36% (20 of 55) Substrates MMP14100% (47 of 47)  98% (46 of 47) 36% (17 of 47) substrates from 1^(st)and 2^(nd) pools MMP14 100% (38 of 38) 100% (38 of 38) 39% (15 of 38)substrates from 1^(st) pool MMP14 100% (9 of 9)  89% (8 of 9) 22% (2 of9) substrates from 2^(nd) pool MMP14 100% (8 of 8) 100% (8 of 8) 38% (3of 8) consensus substrates Combined Total 100% (71 of 71)  99% (70 of71) 42% (30 of 71) MMP9 and MMP14

Example 14 In Vivo Efficacy and In Situ Activation of ActivatableAntibodies Comprising a MMP Substrate

This Example demonstrates that activatable antibodies comprising MMPsubstrates of the embodiments are efficacious in vivo. This Example alsodemonstrates that such activatable antibodies are activatable in an insitu imaging assay, such as that described in International PublicationNo. WO 2014/107559, published 10 Jul. 2014, the contents of which arehereby incorporated by reference in their entirety. Six activatableantibodies comprising different MMP substrates (one MMP9-selected andfive MMP14-selected) of the embodiments were administered at 10 mg/kg or12.5 mg/kg to H292 xenograft tumor-bearing (lung cancer) mice. All sixactivatable antibodies also comprised the masking moiety comprising theamino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 160) and anti-EGFRantibody C225v5 antibody comprising a light chain (SEQ ID NO: 59) and aheavy chain (SEQ ID NO: 56). The configuration of the light chain of theactivatable antibody was masking moiety—MMP substrate—light chain ofC225v5. All six activatable antibodies demonstrated tumor growthinhibition ranging from 22% to 81% as measured by mean % Δ inhibition.Mean % Δ inhibition is calculated as(mean(C)−mean(C0))−(mean(T)−mean(T0))/(mean(C)−mean(C0))*100%, wherein Tis the current test group value, T0 is the current test group initialvalue, C is the control group value, and C0 is the control group initialvalue. The EGFR antibody cetuximab demonstrated 96-98% inhibition inthis study.

The same six activatable antibodies were submitted to in situ imagingassays of H292 tumor tissue, using the conditions described in theexamples of WO 2014/107559. All six activatable antibodies wereactivated, demonstrating that all six MMP substrates were cleaved andthe released antibodies bound to EGFR on the tumor tissue. The stainingsignals ranged from 15% to 65% of the IHC signal intensity of cetuximab.In general, the percentage of activation of each activatable antibodydemonstrated a positive correlation with the efficacy that activatableantibody demonstrated in the H292 mouse model.

Tissue from ten triple negative breast cancer patients was submitted toin situ imaging using an anti-Jagged activatable antibody (e.g., ananti-Jagged activatable antibody cited in International Publication No.WO 2013/192550, published 27 Dec. 2013, the contents of which are herebyincorporated by reference in their entirety) comprising an MMP14-selected substrate under the conditions described in the examples ofWO 2014/107559. Nine of the ten tissue samples demonstrated activatableantibody activation staining scores ranging from 15% to 100% as comparedto the IHC signal intensity of cetuximab: Eight of the ten tissuesamples demonstrated activatable antibody activation staining scoresranging from 30% to 100% as compared to the IHC signal intensity ofcetuximab.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following.

What is claimed:
 1. An isolated polypeptide comprising a cleavablemoiety (CM) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 353-363, 372-375, 376-378, 395-401, 411-419,426-433, 437-449, 454-456, 459-469, 475-482, 487-495, 318-323, 325-327,330-335, 341-347, 14-33, and 159, wherein the cleavable moiety is asubstrate for a protease.
 2. The isolated polypeptide of claim 1,wherein the CM comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 353-363, 372-375, 376-378, 395-401, 411-419,426-433, 437-449, 454-456, 459-469, 475-482, and 487-495.
 3. Theisolated polypeptide of claim 1, wherein the CM comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 318-323,325-327, 330-335, and 341-347.
 4. The isolated polypeptide of claim 1,wherein the CM comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14-33, and
 159. 5. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 353-363, wherein the cleavablemoiety is a substrate for a protease.
 6. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 372-375, wherein the cleavablemoiety is a substrate for a protease.
 7. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 376-378, wherein the cleavablemoiety is a substrate for a protease.
 8. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 395-401, wherein the cleavablemoiety is a substrate for a protease.
 9. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 411-419, wherein the cleavablemoiety is a substrate for a protease.
 10. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 426-433, wherein the cleavablemoiety is a substrate for a protease.
 11. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 437-449, wherein the cleavablemoiety is a substrate for a protease.
 12. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 454-456, wherein the cleavablemoiety is a substrate for a protease.
 13. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 459-469, wherein the cleavablemoiety is a substrate for a protease.
 14. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 475-482, wherein the cleavablemoiety is a substrate for a protease.
 15. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 487-495, wherein the cleavablemoiety is a substrate for a protease.
 16. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 318-323, wherein the cleavablemoiety is a substrate for a protease.
 17. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 325-327, wherein the cleavablemoiety is a substrate for a protease.
 18. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 330-335, wherein the cleavablemoiety is a substrate for a protease.
 19. The isolated polypeptide ofclaim 1, wherein the CM comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 341-347, wherein the cleavablemoiety is a substrate for a protease.
 20. An isolated polypeptidecomprising a cleavable moiety (CM) comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 364-370, 379-393,402-409, 420-424, 434, 435, 450-452, 457, 470-472, 474, and 483, whereinthe cleavable moiety is a substrate for a protease.
 21. An isolatedpolypeptide comprising a cleavable moiety (CM) comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 328, 336-339,and 348-351, wherein the cleavable moiety is a substrate for a protease.22. The isolated polypeptide of claim 1, wherein the CM is cleaved by atleast one matrix metalloprotease (MMP).
 23. The isolated polypeptide ofclaim 22, wherein the CM is cleaved by at least one of MMP9 or MMP14.24. The isolated polypeptide of claim 2, wherein the CM is cleaved by atleast MMP14.
 25. The isolated polypeptide of claim 3, wherein the CM iscleaved by at least MMP9.
 26. The isolated polypeptide of claim 1,wherein the polypeptide comprises an antibody or antigen bindingfragment thereof (AB) that binds a target.
 27. The isolated polypeptideof claim 26, wherein the CM is a substrate for a protease that isco-localized in a tissue with the target.
 28. The isolated polypeptideof claim 26, wherein the antigen binding fragment thereof is selectedfrom the group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv,a scab, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody.
 29. The isolated polypeptide of claim 26, whereinthe AB is linked to the CM.
 30. The isolated polypeptide of claim 29,wherein the AB is linked directly to the CM.
 31. The isolatedpolypeptide of claim 29, wherein the AB is linked to the CM via alinking peptide.
 32. The isolated polypeptide of claim 26, wherein theisolated polypeptide comprises a masking moiety (MM), wherein the MM hasan equilibrium dissociation constant for binding to the AB that isgreater than the equilibrium dissociation constant of the AB for bindingto the target.
 33. The isolated polypeptide of claim 32, wherein the MMis a polypeptide of no more than 40 amino acids in length.
 34. Theisolated polypeptide of claim 32, wherein the MM is linked to the CMsuch that the isolated polypeptide in an uncleaved state comprises thestructural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM.
 35. The isolated polypeptide of claim 34, whereinthe isolated polypeptide comprises a linking peptide between the MM andthe CM.
 36. The isolated polypeptide of claim 34, wherein the isolatedpolypeptide comprises a linking peptide between the CM and the AB. 37.The isolated polypeptide of claim 34, wherein the isolated polypeptidecomprises a first linking peptide (LP1) and a second linking peptide(LP2), and wherein the isolated polypeptide has the structuralarrangement from N-terminus to C-terminus as follows in the uncleavedstate: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
 38. The isolatedpolypeptide of claim 37, wherein the two linking peptides need not beidentical to each other.
 39. The isolated polypeptide of claim 37,wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids inlength.
 40. The isolated polypeptide of claim 32, wherein the amino acidsequence of the MM is different from that of the target and is no morethan 50% identical to the amino acid sequence of a natural bindingpartner of the AB.
 41. The isolated polypeptide of claim 32, wherein theMM does not interfere or compete with the AB for binding to the targetin a cleaved state.